WO2019129086A1 - 一种双向激活共刺激分子受体及其用途 - Google Patents
一种双向激活共刺激分子受体及其用途 Download PDFInfo
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- C12N2510/00—Genetically modified cells
Definitions
- the invention belongs to the field of cell biology and immunology, and relates to a bidirectional activation costimulatory molecule receptor, and the use thereof for modifying a T cell modified by the receptor for treating a malignant tumor.
- Adoptive cell therapy is the process of returning treated autologous or allogeneic immune cells (mainly autologous cells) to tumor patients, directly killing tumor cells, or killing tumor cells by stimulating the body's immune response.
- the purpose of treatment The current tumor adoptive cell therapy has developed rapidly and achieved very good therapeutic effects in the clinical treatment of various malignant tumors (Nature.2016; Jun16; 534(7607): 396-401); (Cell.2016 Oct 6; 167(2 ): 405-418.e13).
- Tumor immune cell therapy is considered to be one of the most promising approaches for the treatment of malignant tumors.
- T cell activation requires stimulation of two signals, two signals related to T cell activation.
- the TCR-CD3 complex on the surface of T cells binds to the antigen peptide-MHC molecule, providing the first signal of T cell activation, determining the killing specificity of T cells; the costimulatory molecules on the surface of T cells (such as CD28) and the corresponding ligands.
- Binding eg, B7 provides a second signal for T cell activation that promotes T cell activation, proliferation, and survival.
- the lack of or the decreased expression of the first signal stimulus source (such as MHC molecule) and the second signal ligand (such as B7) of the tumor cell cannot effectively provide a signal related to T cell activation, thereby failing to activate the T cell immune response.
- the widespread activation of T cell costimulatory molecules may have strong toxic side effects.
- Chimeric antigen receptors activate the intracellular signal CD3 ⁇ or Fc ⁇ RI ⁇ by ITAM (immunoreceptor tyrosine-based activation) by specifically recognizing the single-chain antibody fragment (scFv) of the tumor antigen. Moments.) Signal transmission.
- ITAM immunoglobulin-associated antigen
- scFv single-chain antibody fragment
- the first-generation CAR receptor lacks the costimulatory signal of T cells, which leads to T cells only exerting transient effects, short time in the body and less secretion of cytokines.
- the second and third generation CARs combine the two signals required for T cell activation, and the second signal CD28 or / and 4-1BB intracellular signal regions are directly linked to the CD3 ⁇ molecule, thereby bypassing the tumor.
- Cells often have a second signal, such as B7, that lacks the barrier that prevents T cells from activating.
- B7 a second signal
- the combination of the first signal and the second signal greatly enhances the activation, proliferation and killing ability of T cells, and the therapeutic effect thereof is greatly increased.
- CD28 and 4-1BB molecules can provide the second. Activate the signal and further enhance the TCR/CD3 signal.
- CAR-T cells can only provide stimulation signals to the modified T cells, lack of bystander function, and can not activate surrounding T cells, resulting in a stronger cluster effect, resulting in a series of activated T cell functions. Cascade reaction.
- DCR Dual Costimulatory Activated Receptor
- the modified T cells can not only activate their own costimulatory molecules through extracellular CD137-activated antibodies, but also activate the intracellular cells of the contacted T cells by contact with surrounding unmodified activated T cells.
- Co-stimulating molecular signals to promote T cell activation, proliferation and survival.
- when it co-modifies T cells with the first generation CAR-T containing the first signal it can produce a strong cluster effect and kill tumor cells.
- the effect of this two-way activation is only limited to the T cells that are in contact with each other, and does not cause strong T cell immunity like the activated antibody injected with CD137, causing potentially serious side effects.
- One aspect of the invention relates to an isolated polypeptide comprising, in order from the N-terminus to the C-terminus, the following elements:
- An alternative signal peptide a polypeptide that activates a costimulatory signaling molecule (eg, an activator of a co-stimulatory signaling molecule or a ligand for a costimulatory signaling molecule), an extracellular hinge region, a transmembrane region, and an intracellular costimulatory signaling molecule .
- a costimulatory signaling molecule eg, an activator of a co-stimulatory signaling molecule or a ligand for a costimulatory signaling molecule
- the polypeptide is characterized by any one, two, three, four or five of the following items (1) to (5):
- the signal peptide is a membrane protein signal peptide; preferably, the signal peptide is one or more selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide; preferably, the signal peptide is a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:1;
- the activating single-chain antibody of the costimulatory signal molecule is selected from any one or more of a CD137-activated single-chain antibody, a CD28-activated single-chain antibody, and a CD40-activated single-chain antibody;
- the ligand of the signal molecule is selected from any one or more of a ligand of CD137, a ligand of CD28, and a ligand of CD40;
- amino acid sequence of the CD137-activated single-chain antibody is as shown in SEQ ID NO: 2;
- amino acid sequence of the CD28-activated single-chain antibody is as shown in SEQ ID NO: 31;
- amino acid sequence of the CD40-activated single-chain antibody is as shown in SEQ ID NO: 55;
- the ligand of CD137 is 4-1BBL;
- the ligand of CD28 is CD80/CD86;
- the ligand of CD40 is CD40L;
- the extracellular hinge region is one or more selected from the group consisting of an IgG4Fc CH2CH3 hinge region, a CD28 hinge region, and a CD8 hinge region;
- it is a CD8 hinge region
- amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO: 3;
- it is an IgG4Fc CH2CH3 hinge region
- amino acid sequence of the IgG4 Fc CH2CH3 hinge region is set forth in SEQ ID NO: 56;
- the transmembrane region is one selected from the group consisting of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region.
- the intracellular costimulatory signal molecule is selected from the group consisting of a CD28 intracellular domain, a CD134/OX40 intracellular domain, a CD137/4-1BB intracellular domain, an LCK intracellular domain, an ICOS intracellular domain, and DAP10.
- One or more of the intracellular domains preferably, the intracellular costimulatory signal molecule is a CD28 intracellular domain and/or a CD137 intracellular domain; preferably, the amino acid of the CD28 intracellular domain
- the sequence is set forth in SEQ ID NO: 5; preferably, the amino acid sequence of the intracellular domain of CD137 is set forth in SEQ ID NO: 6.
- the polypeptide in order from the N-terminus to the C-terminus, comprises the following elements:
- An alternative CD8 signal peptide a CD137 activated single chain antibody, a CD8 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and/or a CD137 intracellular domain;
- CD8 signal peptide CD40 activated single chain antibody, IgG4 Fc CH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular domain and/or CD137 intracellular domain.
- polypeptide is as shown in Figures 1A-1 through 1D-1.
- polypeptide is as shown in Figures 1A-2 to 1D-2.
- polypeptide is as shown in Figures 1A-3 to 1D-3.
- the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO: 7 to SEQ ID NO: 14;
- SEQ ID NO: 32 to SEQ ID NO: 39 are shown in any one of the sequences.
- SEQ ID NO: 57 to SEQ ID NO: 64 are shown in any of the sequences.
- Another aspect of the invention relates to an isolated polynucleotide encoding the isolated polypeptide of any of the invention; preferably, the sequence of the isolated polynucleotide is SEQ ID NO: 15 or SEQ ID NO: 22 is shown in any sequence;
- SEQ ID NO: 65 to SEQ ID NO: 72 are shown in any of the sequences.
- a further aspect of the invention relates to a nucleic acid construct comprising a polynucleotide of the invention.
- a further aspect of the present invention relates to a recombinant vector comprising the polynucleotide of the present invention or the nucleic acid construct of the present invention; preferably, the recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant viral vector;
- the recombinant expression vector is a recombinant transposon vector; preferably, the transposon vector contains a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty; preferably, the recombination
- the expression vector is a recombinant vector obtained by recombining the polynucleotide of the present invention and the PS328b vector.
- a further aspect of the invention relates to a recombinant vector combination comprising a first recombinant vector and a second recombinant vector, wherein:
- the first recombinant vector is a recombinant vector of the present invention
- the second recombinant vector contains a coding sequence for a first generation chimeric antigen receptor; preferably, the first generation chimeric antigen receptor is a first generation chimeric antigen targeted to mesothelin, Mucl or EGFR
- the amino acid sequence of the first generation chimeric antigen receptor is as set forth in SEQ ID NO: 23, SEQ ID NO: 48 or SEQ ID NO: 73; preferably, the first generation chimeric antigen
- the nucleic acid sequence of the receptor is as set forth in SEQ ID NO: 24, SEQ ID NO: 49 or SEQ ID NO: 74;
- the second recombinant vector is a recombinant PNB328 vector.
- first and second in the above “first recombination vector” and “second recombination vector” are merely for the purpose of distinction and do not have the meaning of order.
- a further aspect of the invention relates to a recombinant host cell, wherein the cell comprises a polynucleotide of the invention, a nucleic acid construct of the invention, a recombinant vector of the invention or a recombinant vector combination of the invention; preferably,
- the recombinant host cell is a recombinant mammalian cell; preferably, the recombinant host cell is a recombinant T cell; preferably, the recombinant T cell is a recombinant peripheral blood mononuclear cell.
- a further aspect of the invention relates to a T cell expressing a polypeptide according to any of the preceding claims, and a first generation chimeric antigen receptor; preferably, the recombinant T cell is recombinant peripheral blood Mononuclear cells; preferably, the first generation chimeric antigen receptor is a first generation chimeric antigen receptor that targets mesothelin, Mucl or EGFR; preferably, the first generation chimeric antigen receptor
- the amino acid sequence is set forth in SEQ ID NO: 23, SEQ ID NO: 48 or SEQ ID NO: 73.
- a further aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising the polypeptide of any one of the present invention, the polynucleotide of the present invention, the nucleic acid construct of the present invention, the recombinant vector of the present invention, and the present invention
- a further aspect of the invention relates to the polypeptide of any one of the invention, the polynucleotide of the invention, the nucleic acid construct of the invention, the recombinant vector of the invention, the recombinant vector combination of the invention, the recombinant host of the invention
- the cancer is a cancer whose surface of the cancer cell abnormally expresses mesothelin, Mucl or EGFR; preferably, the cancer Selected from: adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.
- a further aspect of the invention relates to the polypeptide of any one of the invention, the polynucleotide of the invention, the nucleic acid construct of the invention, the recombinant vector of the invention, the recombinant vector combination of the invention, the recombinant host of the invention
- a cell or a T cell of the present invention for the preparation of a medicament for inhibiting cancer cells
- the cancer cell is a cancer cell having abnormal expression of mesothelin, Mucl or EGFR on a cell surface
- the cancer cell is selected from the group consisting of Cancer cells of the following cancers: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.
- a further aspect of the invention relates to a method of inhibiting cancer cells in vivo or in vitro, comprising administering to a cancer cell an effective amount of a polypeptide of any of the invention, a polynucleotide of the invention, a nucleic acid of the invention a recombinant vector of the present invention, a recombinant vector combination of the present invention, a recombinant host cell of the present invention or a T cell of the present invention; preferably, the cancer cell is a cell surface abnormally expressing mesothelin, Mucl or EGFR Cancer cells; preferably, the cancer cells are selected from cancer cells of the following cancers: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreas Cancer or prostate cancer.
- a further aspect of the invention relates to a method of treating and/or preventing cancer comprising administering to a subject in need thereof an effective amount of a polypeptide of any of the invention, a polynucleotide of the invention, the invention a nucleic acid construct, a recombinant vector of the present invention, a recombinant vector combination of the present invention, a recombinant host cell of the present invention or a T cell of the present invention; preferably, the cancer has abnormal expression of mesothelin on its cancer cell surface, Cancer of Muc1 or EGFR; preferably, the cancer is selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.
- a further aspect of the invention relates to the polypeptide of any one of the invention, the polynucleotide of the invention, the nucleic acid construct of the invention, the recombinant vector of the invention, the recombinant vector combination of the invention, the recombinant host of the invention
- a further aspect of the invention relates to a method of promoting cytokine secretion of T cells in vivo or in vitro, comprising applying T cells in an effective amount of a polypeptide of any of the invention, a polynucleotide of the invention, a nucleic acid construct of the present invention, a recombinant vector of the present invention, a recombinant vector combination of the present invention, a recombinant host cell of the present invention or a T cell of the present invention; wherein the cytokine is selected from the group consisting of IL-2 and IL-4 One or more of IL-6, IL-10, TNF- ⁇ and IFN- ⁇ .
- the term “isolated” or “isolated” refers to that obtained by artificial means from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
- the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
- vector means a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
- a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
- the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
- Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
- Phage such as lambda phage or M13 phage and animal virus.
- Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
- a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
- the term "host cell” means a cell which can be used for introduction into a vector, which includes, but is not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus, such as S2 fruit. Fly cells such as fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
- a prokaryotic cell such as Escherichia coli or Bacillus subtilis
- a fungal cell such as a yeast cell or an Aspergillus, such as S2 fruit.
- Fly cells such as fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
- chimeric antigen receptor is an artificially engineered receptor capable of anchoring a specific molecule (such as an antibody) that recognizes a tumor antigen to an immune cell (such as a T cell), so that the immune cell recognizes the tumor antigen or Viral antigens and cells that kill tumor cells or virus infection.
- a specific molecule such as an antibody
- an immune cell such as a T cell
- CD137 the official ID number of the NCBI gene bank is 3604, expressed in T cells, and promotes proliferation and activation of T cells.
- it is often used as an intracellular costimulatory signal to enhance the activation and proliferation of T cells.
- CD28 the official ID number of the NCBI gene bank is 940, expressed in T cells, and promotes proliferation and activation of T cells.
- CD28 the official ID number of the NCBI gene bank is 940, expressed in T cells, and promotes proliferation and activation of T cells.
- it is often used as an intracellular costimulatory signal to enhance the activation and proliferation of T cells.
- CD40 the official ID number of the NCBI gene bank is 958, expressed in T cells, and promotes proliferation and activation of T cells.
- CD40 the official ID number of the NCBI gene bank is 958, expressed in T cells, and promotes proliferation and activation of T cells.
- it is often used as an intracellular costimulatory signal to enhance the activation and proliferation of T cells.
- single-chain antibody variable fragment refers to an antibody fragment having the ability to bind antigen by linking the amino acid sequence of the VL region of the antibody and the amino acid sequence of the VH region via Linker.
- V L and V H domains by a linker makes it possible to produce a single polypeptide chain pair to form monovalent molecules (see, e.g., Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl .Acad.Sci. USA 85: 5879-5883 (1988)).
- Such scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
- Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
- a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
- Other linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol.
- T cell activation-associated signal means that the two signals required for T cell activation, that is, the T cell surface TCR-CD3 complex and the antigen peptide-MHC molecule, provide the first signal for T cell activation, Determining the kill specificity of T cells; co-stimulatory molecules on the surface of T cells (such as CD28) bind to the corresponding ligand (such as B7), providing a second signal of T cell activation, promoting T cell activation, proliferation and survival.
- the immunoreceptor tyrosine activating motif is a tyrosine activating motif of CD3 ⁇ and/or Fc ⁇ RI ⁇ ; preferably, the immunoreceptor tyrosine activating motif is a CD3 ⁇ tyrosine activating group
- the amino acid sequence of the sequence is shown in SEQ ID NO: 25.
- co-stimulating molecule in the present invention means some adhesion molecules on the surface of an immune cell, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., activated by binding to its ligand.
- the second signal of immune cells enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
- PB transposon is an abbreviation for Piggybac.
- a transposon is a mobile genetic factor.
- a DNA sequence can be replicated or fragmented separately from the in situ, cyclized and inserted into another site, and the subsequent genes are regulated. This process is called transposition. Due to the function of the transposon on the vector, meso G1 CAR and 137DCR are integrated into the T cell genome.
- Antibodies are classified into activated and blocked antibodies.
- the term "extracellular activated antibody” binds an antibody to the surface of a cell membrane and binds to a site of action of a cell surface molecule (ie, a ligand and a receptor binding site) to promote a cell biological function.
- CD137 extracellular activated antibody because CD137 molecule is present on the surface of most T cells, is considered to be a T cell-specific surface molecule, CD137 extracellular activated antibody can effectively recognize and activate CD137 molecular signal, generate a second signal, CD137 can replace the second signal of APC.
- the term "bystander function" means that when a tumor cell or a virus-infected cell, a single CAR-T cell can only activate the second signal of the self cell, and cannot further activate the peripheral T cell function, resulting in surrounding T cells. Can not cause a series of activated T cell function.
- the term "cluster effect” means that a single modified T cell can continuously recruit and activate surrounding unactivated T cells, and activate peripheral T cell downstream signaling pathways, causing activation and proliferation of multiple T cell levels. .
- the term "mesothelin” is also known as MSLN, meso, mesothelin, and the official ID number of the NCBI gene bank is 10232. It was originally synthesized as a 69 kDa cell surface protein. During the maturation process, it is broken into two segments by the action of furin. The C-terminal 40 kDa fragment is anchored on the membrane, and the N-terminal 32 kDa fragment is released in a dissolved form, called megakaryocyte synergistic factor (MPF). The so-called mesothelin refers to a fragment anchored to a membrane.
- MMF megakaryocyte synergistic factor
- mesothelin is overexpressed in various malignant tumors such as pancreatic cancer, mesothelioma, ovarian cancer, and lung adenocarcinoma, and is a promising target for cell therapy.
- the full-length mesothelin protein can be divided into three segments, Region I (296-390), II (391–486) and III (487–598).
- Muc1 is also called mucin, mucins, and the official ID number of the NCBI gene library is 4582.
- Muc1 is a kind of high molecular weight (>200kD) type I transmembrane glycoprotein (mostly linked by O/glycosidic bond to Ser/Thr on the polypeptide backbone), which is normally expressed mainly in epithelial cells in various tissues and organs.
- the lumen or glandular surface is apical and polar.
- Muc1 protein can be abnormally expressed on the surface of tumor cells, and the expression level is more than 100 times that of normal. Moreover, its polarity distribution on the cell surface is lost and can be evenly distributed throughout the cell surface.
- due to incomplete glycosylation the structure of the Muc1 protein has also changed, and new sugar chains and peptide epitopes have emerged.
- EGFR also known as epidermal growth factor receptor, ErbB-1 or HER1, epidermal growth factor receptor
- EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc.
- EGFR signaling pathway plays an important role in the physiological processes such as cell growth, proliferation and differentiation.
- EGFR is involved in tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis.
- Overexpression of EGFR plays an important role in the progression of malignant tumors, such as glial cell carcinoma, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.
- the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient which is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is It is 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 adjusting agents, surfactants, adjuvants, ionic strength enhancers.
- 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.
- an effective amount means an amount sufficient to obtain or at least partially obtain a desired effect.
- an effective amount to prevent a disease refers to an amount sufficient to prevent, prevent, or delay the onset of a disease (eg, a tumor); treating an effective amount of the disease means sufficient to cure or at least partially arrest a patient already suffering from the disease.
- the amount of disease and its complications Determination of such an effective amount is well within the capabilities of those skilled in the art.
- the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
- the subject may be a mammal, such as a human.
- the CD137 bi-directional costimulatory molecule activating receptor and the mesothelin-modified chimeric antigen receptor-modified T cell can specifically kill tumor cell lines with high expression of mesothelin, and is superior to mesothelin first generation.
- the second generation of CAR-T has little or no killing effect on tumor cell lines that are not expressed, and has high efficiency and high specificity.
- the present invention can maintain the first-generation and second-generation CAR efficacy, and the CD137 bi-directional costimulatory molecule-activated receptor-activated T cell can activate the second signal of the self-cell T cell, and the stronger the tumor-specific antigen is, the first The stronger the activation of the signal CD3 ⁇ , the stronger the second signal associated with the activation of T cells by the extracellular activated antibody of CD137, which accumulates around the tumor and continuously recruits and activates surrounding unactivated T cells and activates T.
- the downstream signaling pathway of the cell causes activation, proliferation and survival of the T cell cascade.
- the effect of this two-way activation is only limited to the T cells that are in contact with each other, and does not cause strong T cell immunity like the activated antibody injected with CD28, causing potentially serious side effects.
- the CD28 bi-directional costimulatory molecule activating receptor provided by the invention combined with the chimeric antigen receptor-modified T cell of Muc1 can specifically kill the tumor cell line with high expression of Muc1, and is superior to the first generation and second generation CAR- of Muc1. T, at the same time, has little or no killing effect on tumor cell lines which are not expressed, and has high efficiency and high specificity.
- the present invention can maintain the therapeutic effect of the first-generation and second-generation CAR, and the CD28 bi-directional costimulatory molecule-activated receptor-activated T cell can activate the second signal of the self-cell T cell, and the stronger the tumor-specific antigen is, the first The stronger the activation of the signal CD3 ⁇ , the stronger the second signal associated with the activation of T cell activation by the extracellular activated antibody of CD28, which accumulates around the tumor and continuously recruits and activates surrounding unactivated T cells and activates T.
- the downstream signaling pathway of the cell causes activation, proliferation and survival of the T cell cascade.
- the CD40 bi-directional costimulatory molecule activating receptor provided by the invention combined with the chimeric antigen receptor-modified T cell of EGFR can specifically kill tumor cell lines with high expression of EGFR, and is superior to EGFR first generation and second generation CAR-. T, at the same time, has little or no killing effect on tumor cell lines which are not expressed, and has high efficiency and high specificity.
- the present invention can maintain the first-generation and second-generation CAR efficacy, and the CD40 bi-directional costimulatory molecule-activated receptor-activated T cell can activate the second signal of the self-cell T cell, and the stronger the tumor-specific antigen is, the first The stronger the activation of the signal CD3 ⁇ , the stronger the second signal associated with the activation of T cell activation by the CD40 extracellular activating antibody, which accumulates around the tumor and continuously recruits and activates surrounding unactivated T cells and activates T.
- the downstream signaling pathway of the cell causes activation, proliferation and survival of the T cell cascade.
- Figure 1A-1 Schematic representation of the structure of CD137 bidirectionally activated costimulatory molecule receptor 137DCR1.
- Figure 1B-1 Schematic representation of the structure of CD137 bidirectionally activated costimulatory molecule receptor 137DCR2.
- Figure 1C-1 Schematic representation of the structure of CD137 bidirectionally activated costimulatory molecule receptor 137DCR3.
- Figure 1D-1 Schematic representation of the structure of CD137 bidirectionally activated costimulatory molecule receptor 137DCR4.
- FIG. 1E-1 Schematic diagram of the meso G1 CAR structure.
- FIG. 1F-1 Schematic diagram of the meso G2 CAR structure.
- Figure 1A-2 Schematic representation of the structure of CD28 bidirectionally activated costimulatory molecule receptor 28DCR1.
- Figure 1B-2 Schematic representation of the structure of CD28 bidirectionally activated costimulatory molecule receptor 28DCR2.
- Figure 1C-2 Schematic representation of the structure of CD28 bidirectionally activated costimulatory molecule receptor 28DCR3.
- Figure 1D-2 Schematic representation of the structure of CD28 bidirectionally activated costimulatory molecule receptor 28DCR4.
- FIG. 1E-2 Schematic diagram of the Muc1 G1 CAR structure.
- FIG. 1F-2 Schematic diagram of the Muc1 G2 CAR structure.
- Figure 1A-3 Schematic representation of the structure of CD40 bidirectionally activated costimulatory molecule receptor 40DCR1.
- Figure 1B-3 Schematic representation of the structure of CD40 bidirectionally activated costimulatory molecule receptor 40DCR2.
- Figure 1C-3 Schematic representation of the structure of CD40 bidirectionally activated costimulatory molecule receptor 40DCR3.
- Figure 1D-3 Schematic representation of the structure of CD40 bidirectionally activated costimulatory molecule receptor 40DCR4.
- Figure 1E-3 Schematic diagram of the EGFR G1 CAR structure.
- Figure 1F-3 Schematic diagram of the EGFR G2 CAR structure.
- Figure 2A-1 Expression of CD3 ⁇ in a bidirectionally activated chimeric antigen receptor mesothelin CAR-T cell.
- the internal reference is GADPH.
- Figure 2B-1 Expression of copy number of 137DCR1, 137DCR2, 137DCR3 in the chimeric antigen receptor mesothelin CAR-T cells.
- Figure 2A-2 Expression of CD3 ⁇ in a bidirectionally activated chimeric antigen receptor Mucl CAR-T cell.
- the internal reference is GADPH.
- Figure 2B-2 Expression of copy number of 28DCR1, 28DCR2, 28DCR3 in the bidirectionally activated chimeric antigen receptor Muc1 CAR-T cells.
- Figure 2A-3 Expression of CD3 ⁇ in a bidirectionally activated chimeric antigen receptor EGFR CAR-T cell.
- the internal reference is GADPH.
- Figure 2B-3 Expression of copy number of 40DCR1, 40DCR2, 40DCR3 in a bidirectionally activated chimeric antigen receptor EGFR CAR-T cell.
- FIG. 3A-1 Electroporation 137 DCR function, Mock T proliferative phenotype.
- the abscissa represents the fluorescence intensity of Hochest positive cells and the ordinate represents the fluorescence intensity of Ki-67 positive cells.
- Ki-67 is channel 6, and Hochest is channel 9.
- the result of co-staining of Ki67 and Hochest, after distinguishing between diploid and tetraploid, Ki67 was used to separate G0 phase resting cells from proliferating cells.
- the first quadrant is the cell undergoing DNA synthesis and division, ie, the cell in the S/G2/M phase; the second quadrant is the preparation phase of the division, ie, the G1 phase.
- FIG. 3B-1 Electroporation of 137DCR1 function, the proliferative phenotype of recombinant cell 137DCR1.
- the abscissa represents the fluorescence intensity of Hochest positive cells and the ordinate represents the fluorescence intensity of Ki-67 positive cells.
- Ki-67 is channel 6, and Hochest is channel 9.
- the result of co-staining of Ki67 and Hochest, after distinguishing between diploid and tetraploid, Ki67 was used to separate G0 phase resting cells from proliferating cells.
- the first quadrant is the cell undergoing DNA synthesis and division, ie, the cell in the S/G2/M phase; the second quadrant is the preparation phase of the division, ie, the G1 phase.
- FIG. 3C-1 Electroporation 137DCR2 function, the proliferative phenotype of recombinant cell 137DCR2.
- the abscissa represents the fluorescence intensity of Hochest positive cells and the ordinate represents the fluorescence intensity of Ki-67 positive cells.
- Ki-67 is channel 6, and Hochest is channel 9.
- the result of co-staining of Ki67 and Hochest, after distinguishing between diploid and tetraploid, Ki67 was used to separate G0 phase resting cells from proliferating cells.
- the first quadrant is the cell undergoing DNA synthesis and division, ie, the cell in the S/G2/M phase; the second quadrant is the preparation phase of the division, ie, the G1 phase.
- FIG. 3D-1 Electroporation of 137DCR3 function, the proliferative phenotype of recombinant cell 137DCR3.
- the abscissa represents the fluorescence intensity of Hochest positive cells and the ordinate represents the fluorescence intensity of Ki-67 positive cells.
- Ki-67 is channel 6, and Hochest is channel 9.
- the result of co-staining of Ki67 and Hochest, after distinguishing between diploid and tetraploid, Ki67 was used to separate G0 phase resting cells from proliferating cells.
- the first quadrant is the cell undergoing DNA synthesis and division, ie, the cell in the S/G2/M phase; the second quadrant is the preparation phase of the division, ie, the G1 phase.
- FIG 3-2 Electrotransfer 28DCR function, recombinant cell 28DCR1/2/3 cell proliferation curve.
- the abscissa represents time (h) and the ordinate represents the number of cells (units).
- FIG. 3-3 Electrotransfer 40DCR function, cell proliferation curve of recombinant cells 40DCR1/2/3 cells.
- the abscissa represents time (h) and the ordinate represents the number of cells (units).
- Figure 4-1 Two-way activation of chimeric antigen receptor mesothelin CAR-T cells, cell proliferation curve.
- Figure 4-2 Two-way activation of the chimeric antigen receptor Mucl CAR-T cells, cell proliferation curve.
- Figure 4-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells, cell proliferation curve.
- FIG. 5A-1 Electroacoustic 137 DCR function, CD137 phenotype of Mock T. Among them, the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 5B-1 Electroporation of 137DCR1 function, CD137 phenotype of recombinant cell 137DCR1.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 5C-1 Electroporation of 137DCR2 function, CD137 phenotype of recombinant cell 137DCR2.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 5D-1 Electroporation of 137DCR3 function, CD137 phenotype of recombinant cell 137DCR3.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 5A-2 Electrical 28DCR function, Mock T CD28 phenotype. Among them, the abscissa is the fluorescence intensity of a single CD28-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5B-2 Electroacoustic 28DCR1 function, CD28 phenotype of recombinant cell 28DCR1.
- the abscissa is the fluorescence intensity of a single CD28-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5C-2 Electrotransfer 28DCR2 function, CD28 phenotype of recombinant cell 28DCR2.
- the abscissa is the fluorescence intensity of a single CD28-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5D-2 Electroacoustic 28DCR3 function, CD28 phenotype of recombinant cell 28DCR3.
- the abscissa is the fluorescence intensity of a single CD28-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5A-3 Electro-rotation 40DCR function, Mock T CD40 phenotype.
- the abscissa is the fluorescence intensity of a single CD40-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5B-3 Electroporation 40DCR1 function, CD40 phenotype of recombinant cell 40DCR1.
- the abscissa is the fluorescence intensity of a single CD40-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5C-3 Electroporation 40DCR2 function, CD40 phenotype of recombinant cell 40DCR2.
- the abscissa is the fluorescence intensity of a single CD40-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- FIG. 5D-3 Electroporation 40DCR3 function, CD40 phenotype of recombinant cell 40DCR3.
- the abscissa is the fluorescence intensity of a single CD40-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6A-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the CD137 phenotype of Mock T. Among them, the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6B-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the CD137 phenotype of recombinant cell meso G1 CAR.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6C-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the CD137 phenotype of recombinant cell meso G2 CAR.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6D-1 The function of electroporation 137DCR in combination with meso G1 CAR, the CD137 phenotype of recombinant cell meso G1 CAR-137DCR1.
- the abscissa is the fluorescence intensity of a single CD137-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6A-2 The function of the electric 28DCR2 combined with the Muc1 G1 CAR, the CD28 phenotype of the Mock T.
- the abscissa is the fluorescence intensity of a single CD28-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 6B-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, CD28 phenotype of recombinant cell Muc1 G1 CAR.
- the abscissa is the fluorescence intensity of a single CD28-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6C-2 The function of electroporation 28DCR2 in combination with Muc1 G1 CAR, CD28 phenotype of recombinant cell Muc1 G2 CAR.
- the abscissa is the fluorescence intensity of a single CD28-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 6D-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, CD28 phenotype of recombinant cell Muc1 G1 CAR-28DCR1.
- the abscissa is the fluorescence intensity of a single CD28-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 6A-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD40 phenotype of Mock T.
- the abscissa is the fluorescence intensity of a single CD40-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 6B-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD40 phenotype of recombinant cell EGFR G1 CAR.
- the abscissa is the fluorescence intensity of a single CD40-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 6C-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD40 phenotype of recombinant cell EGFR G2 CAR.
- the abscissa is the fluorescence intensity of a single CD40-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 6D-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD40 phenotype of recombinant cell EGFR G1 CAR-40DCR1.
- the abscissa is the fluorescence intensity of a single CD40-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7A-1 The function of the electric 137DCR2 combined with the meso G1 CAR, the CD45RO phenotype of the Mock T. Among them, the abscissa is the fluorescence intensity of a single CD45RO-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 7B-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the CD45RO phenotype of recombinant cell meso G1 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 7C-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the CD45RO phenotype of recombinant cell meso G2 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7D-1 The function of electroporation 137DCR in combination with meso G1 CAR, the CD45RO phenotype of recombinant cell meso G1 CAR-137DCR1.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 7A-2 The function of the electric 28DCR2 combined with the Muc1 G1 CAR, the CD45RO phenotype of the Mock T.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7B-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, the CD45RO phenotype of recombinant cell Muc1 G1 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7C-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, the CD45RO phenotype of recombinant cell Muc1 G2 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7D-2 The function of electroporation 28DCR in combination with Muc1 G1 CAR, CD45RO phenotype of recombinant cell Muc1 G1 CAR-28DCR1.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell, and the ordinate is the number of cells with different fluorescence intensities.
- Figure 7A-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD45RO phenotype of Mock T.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7B-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD45RO phenotype of recombinant cell EGFR G1 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7C-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD45RO phenotype of recombinant cell EGFR G2 CAR.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 7D-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the CD45RO phenotype of recombinant cell EGFR G1 CAR-40DCR1.
- the abscissa is the fluorescence intensity of a single CD45RO-positive cell
- the ordinate is the number of cells with different fluorescence intensities.
- Figure 8A-1 The function of the electric 137DCR2 combined with the meso G1 CAR, the memory T phenotype of the Mock T.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8B-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the memory T phenotype of recombinant cell meso G1 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8C-1 The function of electroporation 137DCR2 in combination with meso G1 CAR, the memory T phenotype of recombinant cell meso G2 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8D-1 The function of electroporation 137DCR in combination with meso G1 CAR, the memory T phenotype of recombinant cell meso G1 CAR-137DCR1.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8A-2 The function of the electric 28DCR2 combined with the Muc1 G1 CAR, the memory T phenotype of the Mock T.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8B-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, the memory T phenotype of recombinant cell Muc1 G1 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8C-2 The function of electrotransformation 28DCR2 in combination with Muc1 G1 CAR, the memory T phenotype of recombinant cell Muc1 G2 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the cell fluorescence intensity of a single CCR7-positive cell.
- Figure 8D-2 The function of electrotransformation 28DCR in combination with Muc1 G1 CAR, the memory T phenotype of recombinant cell Muc1 G1CAR-28DCR1.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8A-3 Function of electroporation 40DCR2 in combination with EGFR G1 CAR, memory T phenotype of Mock T.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8B-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the memory T phenotype of recombinant cell EGFR G1 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8C-3 The function of electroporation 40DCR2 in combination with EGFR G1 CAR, the memory T phenotype of recombinant cell EGFR G2 CAR.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 8D-3 The function of electroporation 40DCR in combination with EGFR G1 CAR, the memory T phenotype of recombinant cell EGFR G1 CAR-40DCR1.
- the abscissa is the fluorescence intensity of a single CD62L-positive cell
- the ordinate is the fluorescence intensity of a cell not positive for a single CCR7.
- Figure 9A-1 Two-way activation of chimeric antigen receptor Mesothelin CAR-T cells in vitro against Hela tumor cell line with a target ratio of 8:1 killing.
- Figure 9B-1 Two-way activation of chimeric antigen receptor Mesothelin CAR-T cells have a 4:1 killing effect on Hela tumor cell lines in vitro.
- Figure 9C-1 Two-way activation of chimeric antigen receptor Mesothelin CAR-T cells in vitro against SK-OV-3 tumor cell line with a target ratio of 8:1 killing.
- Figure 9D-1 Two-way activation of chimeric antigen receptor Mesothelin CAR-T cells have a 4:1 killing effect on SK-OV-3 tumor cell lines in vitro.
- Figure 9A-2 Two-way activation of chimeric antigen receptor Mucl CAR-T cells in vitro against MCF7 tumor cell line with a target ratio of 8:1 killing.
- Figure 9B-2 Two-way activation of chimeric antigen receptor Mucl CAR-T cells have a 4:1 killing effect on MCF7 tumor cell lines in vitro.
- Figure 9C-2 Two-way activation of the chimeric antigen receptor Muc1 CAR-T cells have a target ratio of 8:1 to A549 tumor cell lines in vitro.
- Figure 9D-2 Two-way activation of the chimeric antigen receptor Muc1 CAR-T cells have a 4:1 killing effect on A549 tumor cell lines in vitro.
- Figure 9A-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells have an 8:1 killing effect on H23 tumor cell lines in vitro.
- Figure 9B-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells have a 4:1 killing effect on H23 tumor cell lines in vitro.
- Figure 9C-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells have an 8:1 killing effect on ASPC-1 tumor cell lines in vitro.
- Figure 9D-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells have a 4:1 killing effect on ASPC-1 tumor cell lines in vitro.
- Figure 10-1 Two-way activation of chimeric antigen receptor mesothelin CAR-T cells stimulated by mesothelin antigen IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cells Factor changes.
- Figure 10-2 Changes in IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cytokines stimulated by Muc1 antigen in a bidirectionally activated chimeric antigen receptor Muc1 CAR-T cells.
- Figure 10-3 Two-way activation of chimeric antigen receptor EGFR CAR-T cells under EGFR antigen stimulation IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cytokines.
- Figure 11-1 The therapeutic effect of two-way activation of chimeric antigen receptor mesothelin CAR-T cells on a transplanted tumor model of ovarian cancer mice.
- Figure 11-2 Therapeutic effect of two-way activation of the chimeric antigen receptor Mucl CAR-T cells on a transplanted tumor model of ovarian cancer mice.
- Figure 11-3 The therapeutic effect of two-way activation of chimeric antigen receptor EGFR CAR-T cells on a transplanted tumor model of ovarian cancer mice.
- CD3 ⁇ tyrosine activation motif (SEQ ID NO: 25)
- Example 1-(1) 5 recombinant plasmids, pNB328-meso CAR G1, pNB328-meso G2 CAR, Construction of PS328b 137DCR1, PS328b 137DCR2 and PS328b 137DCR3
- the gene for the synthesis of 137DCR1 (SEQ ID NO: 15), the gene for 137DCR2 (SEQ ID NO: 16), the gene for 137DCR3 (SEQ ID NO: 17), the meso G1 CAR gene (SEQ ID NO: 24), and meso
- the G2 CAR gene (SEQ ID NO: 26) has a structural schematic diagram as shown in Figures 1A-1, 1B-1, 1C-1, 1E-1 and 1F-1, respectively.
- the synthesized 5 genes were separately inserted into the PNB328 vector and the PS328b vector, between the EcoRI and SalI cleavage sites.
- the pNB328 vector contains an EF1 ⁇ promoter, a PB transposon, and the like, and the construction of the pNB328 vector is described in Example 2 of WO2017054647A1.
- PS328b is a synthetic sequence synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO:27.
- the constructed recombinant plasmids were named pNB328-meso G1 CAR plasmid, pNB328-meso G2 CAR plasmid, PS328b 137DCR1 plasmid, PS328b 137DCR2 plasmid and PS328b 137DCR3 plasmid, respectively.
- the constructed recombinant plasmid can carry the foreign gene into the genome of the host cell, respectively.
- Example 1-(2) Construction of 5 recombinant plasmids, recombinant plasmid pNB328-Muc1 G1 CAR, pNB328-Muc1 G2 CAR, PS328b 28DCR1, PS328b 28DCR2 and PS328b 28DCR3
- the G2 CAR gene (SEQ ID NO: 50) has a structural schematic diagram as shown in Figures 1A-2, 1B-2, 1C-2, 1E-2 and 1F-2, respectively.
- the synthesized 5 genes were separately inserted into the PNB328 vector and the PS328b vector, between the EcoRI and SalI cleavage sites.
- the pNB328 vector contains an EF1 ⁇ promoter, a PB transposon, and the like, and the construction of the pNB328 vector is described in Example 2 of WO2017054647A1.
- PS328b is a synthetic sequence synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO:27.
- the constructed recombinant plasmids were designated as pNB328-Muc1 G1 CAR plasmid, pNB328-Muc1 G2 CAR plasmid, PS328b 28DCR1 plasmid, PS328b 28DCR2 plasmid and PS328b 28DCR3 plasmid, respectively.
- the constructed recombinant plasmid can carry the foreign gene into the genome of the host cell, respectively.
- Example 1-(3) 5 recombinant plasmids, namely pNB328-EGFR G1 CAR, pNB328-EGFR G2 CAR, Construction of PS328b 40DCR1, PS328b 40DCR2 and PS328b 40DCR3
- the pNB328 vector contains an EF1 ⁇ promoter, a PB transposon, and the like, and the construction of the pNB328 vector is described in Example 2 of WO2017054647A1.
- PS328b is a synthetic sequence synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO:27.
- the constructed recombinant plasmids were designated as pNB328-EGFR G1 CAR plasmid, pNB328-EGFR G2 CAR plasmid, PS328b 40DCR1 plasmid, PS328b 40DCR2 plasmid and PS328b 40DCR3 plasmid, respectively.
- the constructed recombinant plasmid can carry the foreign gene into the genome of the host cell, respectively.
- PBMCs Peripheral blood mononuclear cells
- the suspended cells were collected into a 15 ml centrifuge tube, centrifuged at 1200 rmp for 3 min, and the supernatant was discarded; physiological saline was added, centrifuged at 1200 rmp for 3 min, physiological saline was discarded, and the procedure of "adding physiological saline, centrifugation at 1200 rmp for 3 min, and abandoning physiological saline" was repeated three times.
- each tube is added proportionally to a total of 100 ⁇ l of electroporation reagents, of which:
- the e tube was added with 4 ⁇ g of PS328b 137DCR3 plasmid and pNB328-meso G1 CAR plasmid.
- AIM-V medium containing 2% FBS well-welld solution
- stimulating factors IL-2 and meso/anti-CD28 37 ° C, 5% CO 2 Incubate for 3-4 days to observe the growth of T cells.
- the recombinant T cells of the gene are named as recombinant cell meso G1 CAR, recombinant cell meso G2 CAR, recombinant cell meso G1 CAR-137DCR1, recombinant cell meso G1 CAR-137DCR2, and recombinant cell meso G1 CAR-137DCR3, recombinant cell 137DCR1, recombinant Cell 137DCR2 and recombinant cell 137DCR3.
- meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR1, meso G1CAR-137DCR2, meso G1 CAR-137DCR3 and Mock-T cells were separately collected and washed twice with physiological saline.
- CD3 ⁇ antibody purchased from abcam
- GAPDH antibody purchased from Beyotime
- HRP goat anti-mouse secondary antibody purchased from Jackson
- Genomic DNA (kit method) of recombinant cells meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR1, meso G1 CAR-137DCR2, meso G1 CAR-137DCR3 and Mock-T were extracted, and the experimental procedure was carried out with reference to the kit. Instructions.
- the concentration of DNA in each recombinant cell was determined, and the expression level of 137DCR gene was detected by real-time quantitative PCR.
- the reaction was: 95 ° C, 15 s; 95 ° C, 5 s; 60 ° C, 15 s. 40 cycles.
- the PCR reaction system (20 ⁇ l) was as follows:
- the primer sequences are as follows:
- CD137-F CGAGTCACCATATCAGTA (SEQ ID NO: 28)
- CD137-R CGAAGTACCAGTCATAATTC (SEQ ID NO: 29)
- Example 2-(2) 9 chimeric antigen receptor-modified T cells, ie, recombinant cells Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR1, Muc1 G1 CAR-28DCR2, Muc1 G1 CAR-28DCR3, 28DCR1 Construction and identification of 28DCR2, 28DCR3 and Mock T
- PBMCs Peripheral blood mononuclear cells
- the suspended cells were collected into a 15 ml centrifuge tube, centrifuged at 1200 rmp for 3 min, and the supernatant was discarded; physiological saline was added, centrifuged at 1200 rmp for 3 min, physiological saline was discarded, and the procedure of "adding physiological saline, centrifugation at 1200 rmp for 3 min, and abandoning physiological saline" was repeated three times.
- each tube is proportionally added to a total of 100 ⁇ l of electroporation reagents, of which:
- the e tube was added with 4 ⁇ g of PS328b 28DCR3 plasmid and pNB328-Muc1 G1 CAR plasmid.
- AIM-V medium containing 2% FBS well-welld solution
- stimulating factors IL-2 and Muc1/anti-CD28 37 ° C, 5% CO 2 Incubate for 3-4 days to observe the growth of T cells.
- the recombinant T cells of the gene are named as recombinant cell Muc1 G1 CAR, recombinant cell Muc1 G2 CAR, recombinant cell Muc1 G1 CAR-28DCR1, recombinant cell Muc1 G1 CAR-28DCR2, and recombinant cell Muc1 G1 CAR-28DCR3, recombinant cell 28DCR1, recombinant Cell 28DCR2 and recombinant cell 28DCR3.
- CD3 ⁇ antibody purchased from abcam
- GAPDH antibody purchased from Beyotime
- HRP goat anti-mouse secondary antibody purchased from Jackson
- Genomic DNA (kit method) of recombinant cells Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR1, Muc1 G1 CAR-28DCR2, Muc1 G1 CAR-28DCR3 and Mock-T were extracted, and the experimental procedure was carried out with reference to the kit. Instructions.
- the concentration of DNA in each recombinant cell was determined, and the expression level of 28DCR gene was detected by real-time quantitative PCR.
- the reaction was: 95 ° C, 15 s; 95 ° C, 5 s; 60 ° C, 15 s. 40 cycles.
- the PCR reaction system (20 ⁇ l) was as follows:
- CD28-R 0.4 ⁇ l
- CD28-probe 0.2 ⁇ l
- the primer sequences are as follows:
- CD28-F GCTTCTGGATACACCTTC (SEQ ID NO: 51)
- CD28-R CCTTGAACTTCTCATTATAGTTAG (SEQ ID NO: 52)
- PBMCs Peripheral blood mononuclear cells
- the suspended cells were collected into a 15 ml centrifuge tube, centrifuged at 1200 rmp for 3 min, and the supernatant was discarded; physiological saline was added, centrifuged at 1200 rmp for 3 min, physiological saline was discarded, and the procedure of "adding physiological saline, centrifugation at 1200 rmp for 3 min, and abandoning physiological saline" was repeated three times.
- each tube is proportionally added to a total of 100 ⁇ l of electroporation reagents, of which:
- the e tube was added with 4 ⁇ g of PS328b 40DCR3 plasmid and pNB328-EGFR G1 CAR plasmid.
- AIM-V medium containing 2% FBS well-welld solution
- stimulating factors IL-2 and EGFR / anti-CD28 37 ° C, 5% CO 2 Incubate for 3-4 days to observe the growth of T cells.
- the recombinant T cells of the gene are named as recombinant cell EGFR G1 CAR, recombinant cell EGFR G2 CAR, recombinant cell EGFR G1 CAR-40DCR1, recombinant cell EGFR G1 CAR-40DCR2, and recombinant cell EGFR G1 CAR-40DCR3, recombinant cell 40DCR1, recombinant Cell 40DCR2 and recombinant cell 40DCR3.
- the above recombinant cells were collected for EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR1, EGFR G1CAR-40DCR2, EGFR G1 CAR-40DCR3 and Mock-T cells, respectively, and washed twice with physiological saline.
- CD3 ⁇ antibody (purchased from abcam), GAPDH antibody (purchased from Beyotime), HRP goat anti-mouse secondary antibody (purchased from Jackson), and the expression of CD3 ⁇ in the five recombinant cells previously constructed were detected by western blot or the like. The result is shown in Figure 2A-3.
- Genomic DNA (kit method) of recombinant cells EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR1, EGFR G1 CAR-40DCR2, EGFR G1 CAR-40DCR3 and Mock-T were extracted, and the experimental procedure was carried out with reference to the kit. Instructions.
- the concentration of DNA in each recombinant cell was determined, and the expression level of 40DCR gene was detected by real-time quantitative PCR.
- the reaction was: 95 ° C, 15 s; 95 ° C, 5 s; 60 ° C, 15 s. 40 cycles.
- the PCR reaction system (20 ⁇ l) was as follows:
- CD40-R 0.4 ⁇ l
- CD40-probe 0.2 ⁇ l
- the primer sequences are as follows:
- CD40-F ACCTCCTGATCTATACTG (SEQ ID NO: 76)
- CD40-R GATGGTGAGAGTGAAATC (SEQ ID NO: 77)
- Taqman 5'FAM-CACTGCCGCTGAACCTTGATG-Trama (SEQ ID NO: 78)
- Example 3-(1) Flow cytometry to detect cell proliferation
- Example 2 (1) Recombinant cells prepared 137DCR1, 137DCR2, 137DCR 3 and Mock T.
- Example 2 (1) Recombinant cells prepared by meso G1 CAR, meso G2 CAR, meso G1 CAR-137 DCR2 and Mock T.
- a 96-well white plate was prepared, and each of the above cells on the 8th day of culture was separately taken, and 100 ⁇ L of cell-containing AIM-V medium was added to each well.
- Example 4-(2) Cell Proliferation Viability Kit Detection Cell Technology Detection of Cell Proliferation Activity
- a 96-well white plate was prepared, and each of the above cells on the 8th day of culture was separately taken, and 100 ⁇ L of cell-containing AIM-V medium was added to each well.
- Figure 3-2 shows that Mock T has the slowest proliferation rate and 28DCR proliferation rate is faster.
- Figure 4-2 shows that Mock T has the slowest proliferation rate, Muc1 G1 CAR has a slower proliferation rate, Muc1 G2 CAR has a faster proliferation rate, and Muc1 G1 CAR-28DCR2 has the fastest proliferation rate.
- a 96-well white plate was prepared, and each of the above cells on the 8th day of culture was separately taken, and 100 ⁇ L of cell-containing AIM-V medium was added to each well.
- FIG. 3-3 shows that Mock T has the slowest proliferation rate and 40DCR proliferation rate is faster.
- Figure 4-3 shows that Mock T has the slowest proliferation rate, EGFR G1 CAR has a slower proliferation rate, EGFR G2 CAR has a faster proliferation rate, and EGFR G1 CAR-40DCR2 has the fastest proliferation rate.
- Example 5-(1) Flow-through detection of mesothelin-antigen stimulated bidirectional costimulatory molecule-activated receptor 137DCR combination Meso G1 CAR-T cell phenotype
- Example 2 (1) Recombinant cells prepared 137DCR1, 137DCR2, 137DCR 3, meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR3 and Mock T.
- the above cells were collected separately, counted, and added to a 1.5 ml EP tube at 1 ⁇ 10 6 cells/tube, washed twice with PBS, centrifuged at 1200 rpm for 5 min, and added 2 ⁇ l of the isotype control antibody IgG1-PE, fluorescent flow antibody.
- IgG1-PE fluorescent flow antibody.
- Figures 5A-1 to 5D-1 show three single-transformed cells of three 137DCR1, 137DCR2, and 137DCR3, and the CD137 phenotype is greatly improved relative to Mock T.
- Figures 6A-1 to 6D-1 show the CD137 phenotype of Mock T, meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR2, and the meso G1 CAR-137DCR2 is greatly improved compared to the other three groups.
- Figures 7A-1 to 7D-1 show the CD45RO phenotype of Mock T, meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR2, indicating the degree of cell activation, which have been activated in large amounts.
- Figures 8A-1 to 8D-1 show the memory T phenotype of Mock T, meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR2, and meso G1 CAR-137DCR2 can promote the formation of memory T compared to the other three groups.
- Example 5-(2) Flow cytometry detection of Mut 1 antigen-stimulated bi-directional costimulatory molecule-activated receptor 28DCR combination Muc1 G1 CAR-T cell phenotype
- the above cells were collected separately, counted, and added to a 1.5 ml EP tube at 1 ⁇ 10 6 cells/tube, washed twice with PBS, centrifuged at 1200 rpm for 5 min, and added 2 ⁇ l of the isotype control antibody IgG1-PE, fluorescent flow antibody.
- IgG1-PE fluorescent flow antibody.
- Figures 5A-2 to 5D-2 show three single-transformed cells of three 28DCR1, 28DCR2, and 28DCR3, and the CD137 phenotype is greatly improved relative to Mock T.
- Figures 6A-2 to 6D-2 show the CD137 phenotypes of Mock T, Muc1 G1 CAR, Muc1 G2 CAR, and Muc1 G1 CAR-28DCR2. Compared with the other three groups, Muc1 G1 CAR-28DCR2 is greatly improved.
- Figures 7A-2 to 7D-2 show the CD45RO phenotype of Mock T, Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR2, indicating the degree of cell activation, which have been activated in large amounts.
- 8A-2 to 8D-2 are memory T phenotypes of Mock T, Muc1 G1 CAR, Muc1 G2 CAR, and Muc1 G1 CAR-28DCR2, and Muc1 G1 CAR-28DCR2 can promote the formation of memory T relative to the other three groups.
- Example 5-(3) Flow cytometry detection of bi-directional costimulatory molecule-activated receptor 40DCR combination under EGFR antigen stimulation EGFR G1 CAR-T cell phenotype
- the above cells were collected separately, counted, and added to a 1.5 ml EP tube at 1 ⁇ 10 6 cells/tube, washed twice with PBS, centrifuged at 1200 rpm for 5 min, and added 2 ⁇ l of the isotype control antibody IgG1-PE, fluorescent flow antibody.
- IgG1-PE fluorescent flow antibody.
- Figures 5A-3 to 5D-3 show three single-transformed cells of three 40DCR1, 40DCR2, and 40DCR3, and the CD137 phenotype is greatly improved relative to Mock T;
- Figures 6A-3 to 6D-3 show the CD137 phenotype of Mock T, EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR2, and the EGFR G1 CAR-40DCR2 is greatly improved compared with the other three groups;
- Figures 7A-3 to 7D-3 show the CD45RO phenotype of Mock T, EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR2, indicating the degree of cell activation, which have been activated in large amounts;
- Figures 8A-3 to 8D-3 show the memory T phenotype of Mock T, EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR2, and EGFR G1 CAR-40DCR2 promotes the formation of memory T relative to the other three groups.
- Example 6-(1) Real-time label-free cell function analyzer detects bidirectional costimulatory molecule-activated receptor 137DCR In vitro killing effect of combined meso G1 CAR-T cells on tumor cells
- Effector cells Recombinant cells meso G1 CAR, meso G2 CAR, meso G1 CAR-137DCR1, meso G1 CAR-137DCR3 and Mock T prepared in Example 2-(1).
- Target cells cervical cancer Hela, ovarian cancer cell SK-OV-3 (both purchased from the American Type Culture Collection ATCC).
- RTCA real-time label-free cell function analyzer
- Target cell plating cervical cancer cell Hela and ovarian cancer cell SK-OV-3 were plated in a plate containing the detection electrode at 10 4 cells/50 ⁇ l per well, and left for a few minutes, until the cells were stabilized, and then placed. Into the instrument, start step 2, culture the cells;
- step 2 After 24 hours of target cell culture, stop step 2, add effector cells, 50 ⁇ l per well, and set the effective target ratio to 8:1, 4: (all tumor cells are 10 4 )1
- the plasmid Mock T cells were used as a control, and step 3 was started. After co-culture for 24 hours, the cell proliferation curve was observed.
- Mock T had the weakest killing effect on tumor cells
- meso G1 CAR had weaker killing effect on tumor cells
- meso G2 CAR had stronger killing effect on tumor cells
- meso G1 CAR-137DCR1 meso G1 CAR-137DCR3 pair Tumor cells have the strongest killing effect.
- Example 6-(2) Real-time label-free cell function analyzer detects bidirectional costimulatory molecule-activated receptor 28DCR In vitro killing effect of combined Muc1 G1 CAR-T cells on tumor cells
- Effector cells Recombinant cells prepared in Example 2-(2), Muc1 G1 CAR, Muc1 G2 CAR, Muc1 G1 CAR-28DCR1, Muc1 G1 CAR-28DCR3, 28DCR1, 28DCR2, and 28DCR3 and Mock T.
- Target cells cervical cancer Hela, ovarian cancer cell SK-OV-3 (both purchased from the American Type Culture Collection ATCC).
- RTCA real-time label-free cell function analyzer
- Target cell plating cervical cancer cell Hela and ovarian cancer cell SK-OV-3 were plated in a plate containing the detection electrode at 10 4 cells/50 ⁇ l per well, and left for a few minutes, until the cells were stabilized, and then placed. Into the instrument, start step 2, culture the cells;
- step 2 After 24 hours of target cell culture, stop step 2, add effector cells, 50 ⁇ l per well, and set the effective target ratio to 8:1, 4: (all tumor cells are 10 4 )1
- the plasmid Mock T cells were used as a control, and step 3 was started. After co-culture for 24 hours, the cell proliferation curve was observed.
- Mock T had the weakest killing effect on tumor cells
- Muc1 G1 CAR had weaker killing effect on tumor cells
- Muc1 G2 CAR had stronger killing effect on tumor cells
- Muc1 G1 CAR-28DCR1 Muc1 G1 CAR-28DCR3 pair Tumor cells have the strongest killing effect.
- Example 6-(3) Real-time label-free cell function analyzer detects bidirectional costimulatory molecule-activated receptor 40DCR In vitro killing effect of EGFR G1 CAR-T cells on tumor cells
- Effector cells Recombinant cells prepared in Example 2-(3) EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR1, EGFR G1 CAR-40DCR3, 40DCR1, 40DCR2, 40DCR3 and Mock T.
- Target cells cervical cancer Hela, ovarian cancer cell SK-OV-3 (both purchased from the American Type Culture Collection ATCC).
- RTCA real-time label-free cell function analyzer
- Target cell plating cervical cancer cell Hela and ovarian cancer cell SK-OV-3 were plated in a plate containing the detection electrode at 10 4 cells/50 ⁇ l per well, and left for a few minutes, until the cells were stabilized, and then placed. Into the instrument, start step 2, culture the cells;
- step 2 After 24 hours of target cell culture, stop step 2, add effector cells, 50 ⁇ l per well, and set the effective target ratio to 8:1, 4: (all tumor cells are 10 4 )1
- the plasmid Mock T cells were used as a control, and step 3 was started. After co-culture for 24 hours, the cell proliferation curve was observed.
- Mock T had the weakest killing effect on tumor cells
- EGFR G1 CAR had weaker killing effect on tumor cells
- EGFR G2 CAR had stronger killing effect on tumor cells.
- EGFR G1 CAR-40DCR1, EGFR G1 CAR-40DCR3 pair Tumor cells have the strongest killing effect.
- Example 7-(1) Flow cytometry detection of meso-CAR-T cytokine secretion under stimulation of mesothelin antigen
- Example 2 (1) Recombinant cells meso G1 CAR, meso G2 CAR, meso G1 CAR-137 DCR1 and Mock T.
- a 96-well plate was coated with 5 ⁇ g/ml of mesothelin antigen, coated overnight at 4° C., washed 3 times with PBS, and 1 ⁇ 10 5 of each sample cell was added, respectively, and the cell supernatant was collected after 24 hours of culture.
- BD TM CBA Human Th1 / Th2Cytokine Kit II Checking meso CAR-T cells by secretion after antigen mesothelin cytokine stimulation.
- the secretion of various cytokines of Mock T is the weakest, the secretion of various cytokines of meso G1 CAR is weak, the secretion of various cytokines of meso G2 CAR is strong, and the secretion of various cytokines of meso G1 CAR-137DCR1 The strongest.
- Example 7-(2) Flow cytometry detection of Muc1 CAR-T cytokine secretion under Muc1 antigen stimulation
- a 96-well plate was coated with 5 ⁇ g/ml of Muc1 antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 ⁇ 10 5 of each sample cell was added, and the cell supernatant was collected after 24 hours of culture.
- BD TM CBA Human Th1 / Th2 Cytokine Kit II detecting Muc1 CAR-T cell cytokine secretion after stimulation by antigen Muc1.
- the secretion of various cytokines in Mock T is the weakest.
- the secretion of various cytokines in Muc1 G1 CAR is weak.
- the secretion of various cytokines in Muc1 G2 CAR is strong.
- the secretion of various cytokines in Muc1 G1 CAR-28DCR1 The strongest.
- Example 7-(3) Flow cytometry detection of EGFR CAR-T cytokine secretion under EGFR antigen stimulation
- the recombinant cells prepared in Example 2-(3) were EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR1 and Mock T.
- the 96-well plate was coated with 5 ⁇ g/ml of EGFR antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 ⁇ 10 5 of each sample cell was added, and the cell supernatant was collected after 24 hours of culture.
- BD TM CBA Human Th1 / Th2 Cytokine Kit II detecting EGFR CAR-T cell cytokine secretion after stimulation by EGFR antigen.
- the secretion of various cytokines of Mock T is the weakest, the secretion of various cytokines of EGFR G1 CAR is weak, the secretion of various cytokines of EGFR G2 CAR is strong, and the secretion of various cytokines of EGFR G1 CAR-40DCR1 The strongest.
- Example 8-(1) Two-way costimulatory molecule-activated receptor 137DCR combined with meso G1 CAR-T cell Internal function experiment
- Example 2 (1) Recombinant cells prepared by meso G1 CAR, meso G2 CAR, meso G1 CAR-137 DCR3 and Mock T.
- Human ovarian cancer cell line SK-OV-3-luc was cultured in vitro, adherent growth cells in logarithmic growth phase were taken, digested with 0.25% trypsin, centrifuged, collected, resuspended in PBS, centrifuged at 1000 rpm for 2 minutes at room temperature. The supernatant was discarded, resuspended in PBS, and the cells were collected by centrifugation, and the cell suspension concentration was adjusted to 5 ⁇ 10 7 /ml.
- SK-OV-3-luc cells were inoculated subcutaneously in the right flank of the mouse, 0.1 ml/mouse. After 10 days of inoculation, tumor size can be observed by a live imager.
- mice were randomly divided into 4 groups, 5 in each group.
- the administration route was direct tail vein injection, each 0.1 ml/only, that is, 5 ⁇ 10 6 positive cells, and the solvent was PBS. Only once.
- mice The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 10 days.
- Mock T was a control group, and the fluorescence intensity of tumor cells was strong.
- Meso G1 CAR was a generation of CAR targeting mesothelin.
- the fluorescence intensity of tumor cells was weakened, indicating a certain therapeutic effect.
- the fluorescence intensity of meso G2 CAR tumor cells was weaker. , indicating a better therapeutic effect, meso G1 CAR-137DCR3 tumor cells have the weakest fluorescence intensity, indicating the best therapeutic effect.
- Example 8-(2) Two-way costimulatory molecule-activated receptor 28DCR combined with Muc1 G1 CAR-T cell Internal function experiment
- Human ovarian cancer cell line SK-OV-3-luc was cultured in vitro, adherent growth cells in logarithmic growth phase were taken, digested with 0.25% trypsin, centrifuged, collected, resuspended in PBS, centrifuged at 1000 rpm for 2 minutes at room temperature. The supernatant was discarded, resuspended in PBS, and the cells were collected by centrifugation, and the cell suspension concentration was adjusted to 5 ⁇ 10 7 /ml.
- SK-OV-3-luc cells were inoculated subcutaneously in the right flank of the mouse, 0.1 ml/mouse. After 10 days of inoculation, tumor size can be observed by a live imager.
- mice were randomly divided into 4 groups, 5 in each group.
- the administration route was direct tail vein injection, each 0.1 ml/only, that is, 5 ⁇ 10 6 positive cells, and the solvent was PBS. Only once.
- mice The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 10 days.
- Mock T was a control group
- the fluorescence intensity of tumor cells was strong
- Muc1 G1 CAR was a generation of CAR targeting Mucl
- the fluorescence intensity of tumor cells was weakened, indicating a certain therapeutic effect.
- the fluorescence intensity of Muc1 G2 CAR tumor cells was weaker, indicating With further good therapeutic effects, the fluorescence intensity of Muc1 G1 CAR-28DCR3 tumor cells was the weakest, indicating the best therapeutic effect.
- Example 8-(3) Bidirectional costimulatory molecule-activated receptor 40DCR combined with EGFR G1 CAR-T cell Internal function experiment
- the recombinant cells prepared in Example 2-(3) were EGFR G1 CAR, EGFR G2 CAR, EGFR G1 CAR-40DCR3 and Mock T.
- Human ovarian cancer cell line SK-OV-3-luc was cultured in vitro, adherent growth cells in logarithmic growth phase were taken, digested with 0.25% trypsin, centrifuged, collected, resuspended in PBS, centrifuged at 1000 rpm for 2 minutes at room temperature. The supernatant was discarded, resuspended in PBS, and the cells were collected by centrifugation, and the cell suspension concentration was adjusted to 5 ⁇ 10 7 /ml.
- SK-OV-3-luc cells were inoculated subcutaneously in the right flank of the mouse, 0.1 ml/mouse. After 10 days of inoculation, tumor size can be observed by a live imager.
- mice were randomly divided into 4 groups, 5 in each group.
- the administration route was direct tail vein injection, each 0.1 ml/only, that is, 5 ⁇ 10 6 positive cells, and the solvent was PBS. Only once.
- mice The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 10 days.
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Abstract
提供一种双向激活共刺激分子受体及其用途。所述双向激活共刺激分子受体从N端到C端依次包括下述元件：可选的信号肽、共刺激信号分子激活型单链抗体、胞外铰链区、跨膜区和胞内共刺激信号分子。当所述双向激活共刺激分子受体与包含第一信号的第一代CAR-T共同修饰T细胞时，可以产生强烈的集群效应，杀伤肿瘤细胞，同时不会引发强烈T细胞免疫，造成潜在严重毒副作用。
Description
本发明属于细胞生物学和免疫学领域，涉及一种双向激活共刺激分子受体，及其用该受体修饰后的T细胞治疗恶性肿瘤的用途。
肿瘤过继细胞治疗(adoptive cell therapy，ACT)是将经处理的自体或异体免疫细胞(主要是自体细胞)回输给肿瘤患者，直接杀伤肿瘤细胞，或者通过激发机体的免疫应答杀伤肿瘤细胞，达到治疗目的。当前肿瘤过继细胞治疗发展迅速，在多种恶性肿瘤的临床治疗中取得了非常好的疗效(Nature.2016；Jun16；534(7607):396-401)；(Cell.2016 Oct 6；167(2):405-418.e13)。肿瘤免疫细胞治疗被认为是最有前景的治疗恶性肿瘤的手段之一。
T细胞活化需要两个信号的刺激，即两种T细胞活化相关信号。其中，T细胞表面TCR-CD3复合体与抗原肽-MHC分子结合，提供T细胞活化的第一信号，决定T细胞的杀伤特异性；T细胞表面的共刺激分子(如CD28)与相应配体(如B7)结合，提供T细胞活化的第二信号，促进T细胞活化、增殖与存活。但肿瘤细胞第一信号刺激源(如MHC分子)与第二信号配体(如B7)等缺乏或表达下降，无法有效提供T细胞活化相关的信号，从而无法激活T细胞免疫反应。而广泛性的激活T细胞共刺激分子，可能带来强烈的毒副作用。
嵌合抗原受体CAR(chimeric antigen receptors，CAR)通过胞外特异性识别肿瘤抗原的单链抗体片段(Single chain Variable Fragment,scFv)，激活胞内信号CD3ζ或FcεRIγ的ITAM(immunoreceptor tyrosine-based activation motifs)信号传递。但是第一代CAR受体缺乏T细胞的共刺激信号，导致T细胞只能发挥瞬间效应，在体内存在时间短、细胞因子分泌少。
第二代与第三代的CAR是将T细胞激活所需的两个信号进行了合并，将第二信号CD28或/和4-1BB细胞内信号区域直接连接到CD3ζ分子，从而绕过了肿瘤细胞通常第二信号如B7等缺乏所引起T细胞不能激活的障碍。第一信号与第二信号合并后，大大提高了对T细胞激活、增殖及杀伤能力，使之疗效大幅度增加，基于目前对T细胞激活机制的理解，CD28、4-1BB分子能够提供第二个激活信号并进一 步加强TCR/CD3信号。
然而，不管何种CAR-T细胞，其只能对改造后的T细胞提供刺激信号，缺乏旁观者功能，并不能激活周围T细胞，产生更强的群集效应，引起一系列激活T细胞功能的级联反应。
发明内容
本发明人经过深入的研究和创造性的劳动，设计了一种双向共刺激分子激活受体(Dual Costimulatory Activated Receptor，DCR)，其通过共刺激信号分子胞外激活型抗体传递T细胞活化相关的第二信号。经修饰后的T细胞，不仅能通过胞外的CD137激活型抗体，激活自身的共刺激分子信号，还能通过与周围未修饰活化的T细胞接触后，激活被接触的T细胞的胞内的共刺激分子信号，促进T细胞活化、增殖与存活。特别是，当其与包含第一信号的第一代CAR-T共同修饰T细胞时，可以产生强烈的集群效应，杀伤肿瘤细胞。另外，这种双向激活的作用仅仅局限在相互接触的T细胞之间，不会像注射CD137的激活型抗体一样，引发强烈T细胞免疫，造成潜在严重毒副作用。
由此提供了下述发明：
本发明的一个方面涉及一种分离的多肽，其从N端到C端依次包括下述元件：
可选的信号肽、激活共刺激信号分子的多肽(例如共刺激信号分子的激活型单链抗体或共刺激信号分子的配体)、胞外铰链区、跨膜区和胞内共刺激信号分子。
在本发明的一个或多个实施方案中，所述的多肽，其特征在于如下的(1)－(5)项中的任意1项、2项、3项、4项或者5项：
(1)所述信号肽为膜蛋白信号肽；优选地，所述信号肽为选自CD8信号肽、CD28信号肽和CD4信号肽中的一种或多种；优选地，所述信号肽是CD8信号肽；优选地，CD8信号肽的氨基酸序列如SEQ ID NO:1所示；
(2)所述共刺激信号分子的激活型单链抗体选自CD137激活型单链抗体、CD28激活型单链抗体和CD40激活型单链抗体中的任意一种或者多种；所述共刺激信号分子的配体选自CD137的配体、CD28的配体和CD40的配体中的任意一种或多种；
优选地，所述CD137激活型单链抗体的氨基酸序列如SEQ ID NO:2所示；
优选地，所述CD28激活型单链抗体的氨基酸序列如SEQ ID NO:31所示；
优选地，所述CD40激活型单链抗体的氨基酸序列如SEQ ID NO:55所示；
优选地，所述CD137的配体是4-1BBL；
优选地，所述CD28的配体是CD80/CD86；
优选地，所述CD40的配体是CD40L；
(3)所述胞外铰链区为选自IgG4Fc CH2CH3铰链区、CD28铰链区和CD8铰链区的一种或多种；
优选地，为CD8铰链区；
优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:3所示；
优选地，为IgG4Fc CH2CH3铰链区；
优选地，所述IgG4Fc CH2CH3铰链区的氨基酸序列如SEQ ID NO:56所示；
(4)所述跨膜区为选自CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种；优选地，所述跨膜区为CD28跨膜区；优选地，所述CD28跨膜区的氨基酸序列如SEQ ID NO:4所示；
(5)所述胞内共刺激信号分子选自CD28胞内结构域、CD134/OX40胞内结构域、CD137/4-1BB胞内结构域、LCK胞内结构域、ICOS胞内结构域和DAP10胞内结构域中的一种或多种；优选地，所述胞内共刺激信号分子为CD28胞内结构域和/或CD137胞内结构域；优选地，所述CD28胞内结构域的氨基酸序列如SEQ ID NO:5所示；优选地，所述CD137胞内结构域的氨基酸序列如SEQ ID NO:6所示。
在本发明的一个或多个实施方案中，所述的多肽，其从N端到C端依次包括下述元件：
可选的CD8信号肽、CD137激活型单链抗体、CD8胞外铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域；
可选的CD8信号肽、CD28激活型单链抗体、CD8胞外铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域；或者
可选的CD8信号肽、CD40激活型单链抗体、IgG4Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域。
在本发明的一个或多个实施方案中，所述的多肽，其如图1A-1至图1D-1所示。
在本发明的一个或多个实施方案中，所述的多肽，其如图1A-2至图1D-2所示。
在本发明的一个或多个实施方案中，所述的多肽，其如图1A-3至图1D-3所示。
在本发明的一个或多个实施方案中，所述的多肽，其氨基酸序列如SEQ ID NO:7 至SEQ ID NO:14中任一序列所示；
SEQ ID NO:32至SEQ ID NO:39中任一序列所示；或者
SEQ ID NO:57至SEQ ID NO:64中任一序列所示。
本发明的另一方面涉及一种分离的多核苷酸，其编码本发明中任一项所述的分离的多肽；优选地，所述分离的多核苷酸的序列如SEQ ID NO:15或SEQ ID NO:22中任一序列所示；
SEQ ID NO:40至SEQ ID NO:47中任一序列所示；或者
SEQ ID NO:65至SEQ ID NO:72中任一序列所示。
本发明的再一方面涉及一种核酸构建体，包含本发明的多核苷酸。
本发明的再一方面涉及一种重组载体，其含有本发明的多核苷酸或者本发明的核酸构建体；优选地，所述重组载体为重组克隆载体、重组真核表达质粒或者重组病毒载体；优选地，所述重组表达载体为重组的转座子载体；优选地，所述转座子载体含有选自piggybac、sleeping beauty、frog prince、Tn5或Ty的转座元件；优选地，所述重组表达载体为本发明的多核苷酸与PS328b载体经重组得到的重组载体。
本发明的再一方面涉及一种重组载体组合，其包含第一重组载体和第二重组载体，其中：
所述第一重组载体为本发明的重组载体，
所述第二重组载体含有第一代嵌合抗原受体的编码序列；优选地，所述第一代嵌合抗原受体为靶向间皮素、Muc1或EGFR的第一代嵌合抗原受体；优选地，所述第一代嵌合抗原受体的氨基酸序列如SEQ ID NO:23、SEQ ID NO:48或SEQ ID NO:73所示；优选地，所述第一代嵌合抗原受体的核酸序列如SEQ ID NO:24、SEQ ID NO:49或SEQ ID NO:74所示；
优选地，所述第二重组载体为重组的PNB328载体。
其中，上述“第一重组载体”和“第二重组载体”中的“第一”和“第二”仅仅是为了指代上的区分，并不具有次序的含义。
本发明的再一方面涉及一种重组宿主细胞，其中，所述细胞含有本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体或者本发明的重组载体组合；优选地，所述重组宿主细胞为重组哺乳动物细胞；优选地，所述重组宿主细胞为重组T细胞； 优选地，所述重组T细胞为重组的外周血单核细胞。
本发明的再一方面涉及一种T细胞，其表达有本发明中任一权项所述的多肽，以及第一代嵌合抗原受体；优选地，所述重组T细胞为重组的外周血单核细胞；优选地，所述第一代嵌合抗原受体为靶向间皮素、Muc1或EGFR的第一代嵌合抗原受体；优选地，所述第一代嵌合抗原受体的氨基酸序列如SEQ ID NO:23、SEQ ID NO:48或SEQ ID NO:73所示。
本发明的再一方面涉及一种药用组合物，其包含本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞；可选地，还包含药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞在制备治疗和/或预防癌症的药物中的用途；优选地，所述癌症为其癌细胞表面异常表达间皮素、Muc1或EGFR的癌症；优选地，所述癌症选自：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
本发明的再一方面涉及本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞在制备抑制癌细胞的药物中的用途；优选地，所述癌细胞为细胞表面异常表达间皮素、Muc1或EGFR的癌细胞；优选地，所述癌细胞选自如下癌症的癌细胞：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
本发明的再一方面涉及一种在体内或体外抑制癌细胞的方法，包括给予癌细胞以有效量的本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞的步骤；优选地，所述癌细胞为细胞表面异常表达间皮素、Muc1或EGFR的癌细胞；优选地，所述癌细胞选自如下癌症的癌细胞：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
本发明的再一方面涉及一种治疗和/或预防癌症的方法，包括给予有需求的受试者 以有效量的本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞的步骤；优选地，所述癌症为其癌细胞表面异常表达间皮素、Muc1或EGFR的癌症；优选地，所述癌症选自：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
本发明的再一方面涉及本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞在制备促进细胞因子分泌的药物中的用途，其中，所述细胞因子选自IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ中的一种或多种。
本发明的再一方面涉及一种在体内或在体外促进T细胞的细胞因子分泌的方法，包括施加T细胞以有效量的本发明中任一项所述的多肽、本发明的多核苷酸、本发明的核酸构建体、本发明的重组载体、本发明的重组载体组合、本发明的重组宿主细胞或者本发明的T细胞的步骤；其中，所述细胞因子选自IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ中的一种或多种。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本发明中，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
本发明中，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来 源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
本发明中，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
本发明中，术语“嵌合抗原受体”是人工改造受体，能够将识别肿瘤抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原或病毒抗原和杀死肿瘤细胞或病毒感染的细胞。
本发明中，术语“CD137”，NCBI基因库的官方ID号为3604，表达于T细胞中，可促进T细胞的增殖和活化。在第二代嵌合型抗原受体修饰的T细胞疗法中，常作为胞内共刺激信号，增强T细胞的活化和增殖能力。
本发明中，术语“CD28”，NCBI基因库的官方ID号为940，表达于T细胞中，可促进T细胞的增殖和活化。在第二代嵌合型抗原受体修饰的T细胞疗法中，常作为胞内共刺激信号，增强T细胞的活化和增殖能力。
本发明中，术语“CD40”，NCBI基因库的官方ID号为958，表达于T细胞中，可促进T细胞的增殖和活化。在第二代嵌合型抗原受体修饰的T细胞疗法中，常作为胞内共刺激信号，增强T细胞的活化和增殖能力。
本发明中，术语“单链抗体”(single-chain antibody variable fragment,scFv)是指由抗体V
L区氨基酸序列和V
H区氨基酸序列经Linker连接而成，具有结合抗原能力的抗体片段。其中V
L和V
H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423-426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。此类scFv分子可具有一般结构：NH
2-V
L-接头-V
H-COOH或NH
2-V
H-接头-V
L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)
4的接头， 但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本发明中，术语“T细胞活化相关信号”是指与T细胞活化所需要两个信号，即T细胞表面TCR-CD3复合体与抗原肽-MHC分子结合，提供T细胞活化的第一信号，决定T细胞的杀伤特异性；T细胞表面的共刺激分子(如CD28)与相应配体(如B7)结合，提供T细胞活化的第二信号，促进T细胞活化、增殖与存活。
本发明中，所述免疫受体酪氨酸活化基序为CD3ζ和/或FcεRIγ的酪氨酸活化基序；优选地，所述免疫受体酪氨酸活化基序为CD3ζ酪氨酸活化基序，其氨基酸序列如SEQ ID NO:25所示。
本发明中术语“共刺激信号分子”(Co-stimulating molecule)是指免疫细胞表面的一些粘附分子，如CD28、CD134/OX40、CD137/4-1BB、CD40等，通过与其配体结合，激活免疫细胞的第二信号，增强免疫细胞的增殖能力及细胞因子的分泌功能，延长活化免疫细胞的存活时间。
本发明中，术语“PB”转座子，是Piggybac的简称。转座子是一段可移动的遗传因子。一段DNA序列可以从原位上单独复制或断裂下来，环化后插入另一位点，并对其后的基因起调控作用，此过程称转座。由于载体上的转座子发挥功能，使meso G1 CAR、137DCR整合入T细胞基因组。
抗体分为激活型和阻断型抗体。本发明中，术语“胞外激活型抗体”是将抗体锚定在细胞膜表面，和细胞表面分子的作用位点(即配体和受体结合部位)结合，促进细胞生物学功能。CD137胞外激活型抗体，由于CD137分子存在于大多数T细胞表面，被认为是一种T细胞特有的表面分子,CD137胞外激活型抗体可有效识别并激活CD137分子信号，产生第二信号，CD137可替代APC的第二信号作用。
本发明中，术语“旁观者功能”是指当肿瘤细胞、病毒感染的细胞时，单个CAR-T细胞，只能激活自身细胞第二信号，并不能进一步激活周围T细胞功能，导致周围T细胞不能引起一系列激活T细胞功能。
本发明中，术语“群集效应”是指单个修饰后的T细胞能够不断招募并激活周围未活化的T细胞，并激活周围T细胞下游信号通路，引起多个T细胞级的活化、增殖等 功能。
本发明中，术语“间皮素”又称MSLN、meso、mesothelin，NCBI基因库的官方ID号为10232。它最初合成的是一个69kDa的细胞表面蛋白。成熟过程中在弗林蛋白酶的作用下断裂成两段，C末端40kDa的片段锚定在膜上，N末端32kDa的片段以溶解的形式释放出来，称为巨核细胞增效因子(MPF)。通常所说的间皮素指的是锚定在膜上的片段。间皮素在胰腺癌、间皮瘤、卵巢癌、肺腺癌等多种恶性肿瘤中过表达，是一个很有前景的细胞治疗靶点。全长间皮素蛋白可分为三段，Region I(296-390)，II(391–486)和III(487–598)。
本发明中，术语“Muc1”又称粘蛋白、mucins，NCBI基因库的官方ID号为4582。Muc1是一类高分子量(>200kD)的Ⅰ型跨膜糖蛋白(多以O-糖苷键与多肽骨架上的Ser/Thr相连)，正常情况下主要表达于多种组织、器官中上皮细胞近管腔或腺腔面，呈顶端表达，极性分布。在肿瘤发生时，Muc1蛋白可在肿瘤细胞表面异常表达，表达量可达正常时的100倍以上。并且，其在细胞表面的极性分布丧失，可在整个细胞表面均匀分布。另外，由于糖基化不全，Muc1蛋白的结构也发生改变，出现新的糖链及肽表位。
本发明中，术语“EGFR”又称表皮生长因子受体、ErbB-1或HER1，epidermal growth factor receptor，NCBI基因库的官方ID号为1956。EGFR广泛分布于哺乳动物上皮细胞、成纤维细胞、胶质细胞、角质细胞等细胞表面，EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥重要的作用。EGFR与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关。EGFR的过表达在恶性肿瘤的演进中起重要作用，胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、乳腺癌等。
本发明中，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
本发明中，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发 生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本发明中，所述受试者可以为哺乳动物，例如人。
发明的有益效果
当其与包含第一信号的第一代CAR-T共同修饰T细胞时，可以产生强烈的集群效应，杀伤肿瘤细胞。同时，这种双向激活的作用仅仅局限在相互接触的T细胞之间，不会像注射CD137的激活型抗体一样，引发强烈T细胞免疫，造成潜在严重毒副作用。本发明提供的CD137双向共刺激分子激活受体联合间皮素的嵌合抗原受体修饰的T细胞，可以特异性杀伤间皮素高表达的肿瘤细胞株，并且优于间皮素第一代、第二代CAR-T，同时对不表达的肿瘤细胞株杀伤作用较小或无，具有高效、高特异性。本发明能在保持第一代、第二代CAR疗效的基础上，CD137双向共刺激分子激活受体活化的T细胞能够激活自身T细胞第二信号，是针对肿瘤特异性抗原越强，第一信号CD3ζ激活也越强，相应地，CD137胞外激活型抗体传递T细胞活化相关的第二信号也越强，聚集在肿瘤局部周围，并且不断招募并激活周围未活化的T细胞，并激活T细胞下游信号通路，引起T细胞级联式的活化、增殖与存活。
当其与包含第一信号的第一代CAR-T共同修饰T细胞时，可以产生强烈的集群效应，杀伤肿瘤细胞。同时，这种双向激活的作用仅仅局限在相互接触的T细胞之间，不会像注射CD28的激活型抗体一样，引发强烈T细胞免疫，造成潜在严重毒副作用。本发明提供的CD28双向共刺激分子激活受体联合Muc1的嵌合抗原受体修饰的T细胞，可以特异性杀伤Muc1高表达的肿瘤细胞株，并且优于Muc1第一代、第二代CAR-T，同时对不表达的肿瘤细胞株杀伤作用较小或无，具有高效、高特异性。本发明能在保持第一代、第二代CAR疗效的基础上，CD28双向共刺激分子激活受体活化的T细胞能够激活自身T细胞第二信号，是针对肿瘤特异性抗原越强，第一信号CD3ζ激活也越强，相应地，CD28胞外激活型抗体传递T细胞活化相关的第二信号也越强，聚集在肿瘤局部周围，并且不断招募并激活周围未活化的T细胞，并激活T细胞下游信号通路，引起T细胞级联式的活化、增殖与存活。
当其与包含第一信号的第一代CAR-T共同修饰T细胞时，可以产生强烈的集群效应，杀伤肿瘤细胞。同时，这种双向激活的作用仅仅局限在相互接触的T细胞之间，不会像注射CD40的激活型抗体一样，引发强烈T细胞免疫，造成潜在严重毒副作用。本发明提供的CD40双向共刺激分子激活受体联合EGFR的嵌合抗原受体修饰的T细胞，可以特异性杀伤EGFR高表达的肿瘤细胞株，并且优于EGFR第一代、第二代CAR-T，同时对不表达的肿瘤细胞株杀伤作用较小或无，具有高效、高特异性。本发明能在保持第一代、第二代CAR疗效的基础上，CD40双向共刺激分子激活受体活化的T细胞能够激活自身T细胞第二信号，是针对肿瘤特异性抗原越强，第一信号CD3ζ激活也越强，相应地，CD40胞外激活型抗体传递T细胞活化相关的第二信号也越强，聚集在肿瘤局部周围，并且不断招募并激活周围未活化的T细胞，并激活T细胞下游信号通路，引起T细胞级联式的活化、增殖与存活。
图1A-1：CD137双向激活共刺激分子受体137DCR1的结构示意图。
图1B-1：CD137双向激活共刺激分子受体137DCR2的结构示意图。
图1C-1：CD137双向激活共刺激分子受体137DCR3的结构示意图。
图1D-1：CD137双向激活共刺激分子受体137DCR 4的结构示意图。
图1E-1：meso G1 CAR结构示意图。
图1F-1：meso G2 CAR结构示意图。
图1A-2：CD28双向激活共刺激分子受体28DCR1的结构示意图。
图1B-2：CD28双向激活共刺激分子受体28DCR2的结构示意图。
图1C-2：CD28双向激活共刺激分子受体28DCR3的结构示意图。
图1D-2：CD28双向激活共刺激分子受体28DCR4的结构示意图。
图1E-2：Muc1 G1 CAR结构示意图。
图1F-2：Muc1 G2 CAR结构示意图。
图1A-3：CD40双向激活共刺激分子受体40DCR1的结构示意图。
图1B-3：CD40双向激活共刺激分子受体40DCR2的结构示意图。
图1C-3：CD40双向激活共刺激分子受体40DCR3的结构示意图。
图1D-3：CD40双向激活共刺激分子受体40DCR4的结构示意图。
图1E-3：EGFR G1 CAR结构示意图。
图1F-3：EGFR G2 CAR结构示意图。
图2A-1：双向激活嵌合抗原受体间皮素CAR-T细胞中CD3ζ的表达情况。内参为GADPH。
图2B-1：双向激活嵌合抗原受体间皮素CAR-T细胞中137DCR1、137DCR2、137DCR3的拷贝数的表达情况。
图2A-2：双向激活嵌合抗原受体Muc1 CAR-T细胞中CD3ζ的表达情况。内参为GADPH。
图2B-2：双向激活嵌合抗原受体Muc1 CAR-T细胞中28DCR1、28DCR2、28DCR3的拷贝数的表达情况。
图2A-3：双向激活嵌合抗原受体EGFR CAR-T细胞中CD3ζ的表达情况。内参为GADPH。
图2B-3：双向激活嵌合抗原受体EGFR CAR-T细胞中40DCR1、40DCR2、40DCR3的拷贝数的表达情况。
图3A-1：电转137DCR功能，Mock T的增殖表型。横坐标代表Hochest阳性的细胞荧光强度，纵坐标代表Ki-67阳性的细胞荧光强度。Ki-67为通道6，Hochest为通道9。Ki67和Hochest共染的结果，在区分二倍体和四倍体后，用Ki67将G0期的静息态细胞和增殖态细胞分离。第一象限是正在进行DNA合成以及分裂的细胞，即处于S/G2/M期的细胞；第二象限是分裂的准备期，即G1期。
图3B-1：电转137DCR1功能，重组细胞137DCR1的增殖表型。横坐标代表Hochest阳性的细胞荧光强度，纵坐标代表Ki-67阳性的细胞荧光强度。Ki-67为通道6，Hochest为通道9。Ki67和Hochest共染的结果，在区分二倍体和四倍体后，用Ki67将G0期的静息态细胞和增殖态细胞分离。第一象限是正在进行DNA合成以及分裂的细胞，即处于S/G2/M期的细胞；第二象限是分裂的准备期，即G1期。
图3C-1：电转137DCR2功能，重组细胞137DCR2的增殖表型。横坐标代表Hochest阳性的细胞荧光强度，纵坐标代表Ki-67阳性的细胞荧光强度。Ki-67为通道6，Hochest为通道9。Ki67和Hochest共染的结果，在区分二倍体和四倍体后，用Ki67将G0期的静息态细胞和增殖态细胞分离。第一象限是正在进行DNA合成以及分裂的细胞，即处于S/G2/M期的细胞；第二象限是分裂的准备期，即G1期。
图3D-1：电转137DCR3功能，重组细胞137DCR3的增殖表型。横坐标代表Hochest阳性的细胞荧光强度，纵坐标代表Ki-67阳性的细胞荧光强度。Ki-67为通道6，Hochest 为通道9。Ki67和Hochest共染的结果，在区分二倍体和四倍体后，用Ki67将G0期的静息态细胞和增殖态细胞分离。第一象限是正在进行DNA合成以及分裂的细胞，即处于S/G2/M期的细胞；第二象限是分裂的准备期，即G1期。
图3-2：电转28DCR功能，重组细胞28DCR1/2/3细胞细胞增殖曲线。横坐标代表时间(h)，纵坐标代表细胞数(个)。
图3-3：电转40DCR功能，重组细胞40DCR1/2/3细胞细胞增殖曲线。横坐标代表时间(h)，纵坐标代表细胞数(个)。
图4-1：双向激活嵌合抗原受体间皮素CAR-T细胞，细胞增殖曲线。
图4-2：双向激活嵌合抗原受体Muc1 CAR-T细胞，细胞增殖曲线。
图4-3：双向激活嵌合抗原受体EGFR CAR-T细胞，细胞增殖曲线。
图5A-1：电转137DCR功能，Mock T的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5B-1：电转137DCR1功能，重组细胞137DCR1的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5C-1：电转137DCR2功能，重组细胞137DCR2的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5D-1：电转137DCR3功能，重组细胞137DCR3的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5A-2：电转28DCR功能，Mock T的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5B-2：电转28DCR1功能，重组细胞28DCR1的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5C-2：电转28DCR2功能，重组细胞28DCR2的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5D-2：电转28DCR3功能，重组细胞28DCR3的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5A-3：电转40DCR功能，Mock T的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5B-3：电转40DCR1功能，重组细胞40DCR1的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5C-3：电转40DCR2功能，重组细胞40DCR2的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图5D-3：电转40DCR3功能，重组细胞40DCR3的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6A-1：电转137DCR2联合meso G1 CAR的功能，Mock T的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6B-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G1 CAR的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6C-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G2 CAR的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6D-1：电转137DCR联合meso G1 CAR的功能，重组细胞meso G1 CAR-137DCR1的CD137表型。其中，横坐标为单个CD137阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6A-2：电转28DCR2联合Muc1 G1 CAR的功能，Mock T的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6B-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G1 CAR的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6C-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G2 CAR的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6D-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G1 CAR-28DCR1的CD28表型。其中，横坐标为单个CD28阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6A-3：电转40DCR2联合EGFR G1 CAR的功能，Mock T的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6B-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光 强度的细胞数。
图6C-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G2 CAR的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图6D-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR-40DCR1的CD40表型。其中，横坐标为单个CD40阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7A-1：电转137DCR2联合meso G1 CAR的功能，Mock T的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7B-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G1 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7C-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G2 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7D-1：电转137DCR联合meso G1 CAR的功能，重组细胞meso G1 CAR-137DCR1的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7A-2：电转28DCR2联合Muc1 G1 CAR的功能，Mock T的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7B-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G1 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7C-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G2 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7D-2：电转28DCR联合Muc1 G1 CAR的功能，重组细胞Muc1 G1 CAR-28DCR1的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7A-3：电转40DCR2联合EGFR G1 CAR的功能，Mock T的CD45RO表型。 其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7B-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7C-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G2 CAR的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图7D-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR-40DCR1的CD45RO表型。其中，横坐标为单个CD45RO阳性的细胞荧光强度，纵坐标为不同荧光强度的细胞数。
图8A-1：电转137DCR2联合meso G1 CAR的功能，Mock T的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8B-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G1 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8C-1：电转137DCR2联合meso G1 CAR的功能，重组细胞meso G2 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8D-1：电转137DCR联合meso G1 CAR的功能，重组细胞meso G1 CAR-137DCR1的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8A-2：电转28DCR2联合Muc1 G1 CAR的功能，Mock T的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8B-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G1 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8C-2：电转28DCR2联合Muc1 G1 CAR的功能，重组细胞Muc1 G2 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7 阳性的细胞荧光强度。
图8D-2：电转28DCR联合Muc1 G1 CAR的功能，重组细胞Muc1 G1CAR-28DCR1的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8A-3：电转40DCR2联合EGFR G1 CAR的功能，Mock T的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8B-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8C-3：电转40DCR2联合EGFR G1 CAR的功能，重组细胞EGFR G2 CAR的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图8D-3：电转40DCR联合EGFR G1 CAR的功能，重组细胞EGFR G1 CAR-40DCR1的记忆T表型。其中，横坐标为单个CD62L阳性的细胞荧光强度，纵坐标为不单个CCR7阳性的细胞荧光强度。
图9A-1：双向激活嵌合抗原受体间皮素CAR-T细胞在体外对Hela肿瘤细胞株效靶比为8:1的杀伤。
图9B-1：双向激活嵌合抗原受体间皮素CAR-T细胞在体外对Hela肿瘤细胞株效靶比为4:1的杀伤。
图9C-1：双向激活嵌合抗原受体间皮素CAR-T细胞在体外对SK-OV-3肿瘤细胞株效靶比为8:1的杀伤。
图9D-1：双向激活嵌合抗原受体间皮素CAR-T细胞在体外对SK-OV-3肿瘤细胞株效靶比为4:1的杀伤。
图9A-2：双向激活嵌合抗原受体Muc1 CAR-T细胞在体外对MCF7肿瘤细胞株效靶比为8:1的杀伤。
图9B-2：双向激活嵌合抗原受体Muc1 CAR-T细胞在体外对MCF7肿瘤细胞株效靶比为4:1的杀伤。
图9C-2：双向激活嵌合抗原受体Muc1 CAR-T细胞在体外对A549肿瘤细胞株效靶比为8:1的杀伤。
图9D-2：双向激活嵌合抗原受体Muc1 CAR-T细胞在体外对A549肿瘤细胞株效靶比为4:1的杀伤。
图9A-3：双向激活嵌合抗原受体EGFR CAR-T细胞在体外对H23肿瘤细胞株效靶比为8:1的杀伤。
图9B-3：双向激活嵌合抗原受体EGFR CAR-T细胞在体外对H23肿瘤细胞株效靶比为4:1的杀伤。
图9C-3：双向激活嵌合抗原受体EGFR CAR-T细胞在体外对ASPC-1肿瘤细胞株效靶比为8:1的杀伤。
图9D-3：双向激活嵌合抗原受体EGFR CAR-T细胞在体外对ASPC-1肿瘤细胞株效靶比为4:1的杀伤。
图10-1：双向激活嵌合抗原受体间皮素CAR-T细胞在间皮素抗原刺激下IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ细胞因子的变化。
图10-2：双向激活嵌合抗原受体Muc1 CAR-T细胞在Muc1抗原刺激下IL-2，IL-4，IL-6,IL-10，TNF-α和IFN-γ细胞因子的变化。
图10-3：双向激活嵌合抗原受体EGFR CAR-T细胞在EGFR抗原刺激下IL-2，IL-4，IL-6,IL-10，TNF-α和IFN-γ细胞因子的变化。
图11-1：双向激活嵌合抗原受体间皮素CAR-T细胞对卵巢癌小鼠移植瘤模型的治疗效果。
图11-2：双向激活嵌合抗原受体Muc1 CAR-T细胞对卵巢癌小鼠移植瘤模型的治疗效果。
图11-3：双向激活嵌合抗原受体EGFR CAR-T细胞对卵巢癌小鼠移植瘤模型的治疗效果。
本发明涉及的部分序列如下：
1.CD8信号肽的氨基酸序列(SEQ ID NO:1)
2.CD137胞外激活型单链抗体的氨基酸序列(SEQ ID NO:2)
3.CD8α铰链区的氨基酸序列(SEQ ID NO:3)
4.CD28跨膜区的氨基酸序列(SEQ ID NO:4)
5.CD28胞内共刺激信号胞内结构域的氨基酸序列(SEQ ID NO:5)
6.CD137胞内共刺激信号胞内结构域的氨基酸序列(SEQ ID NO:6)
7. 137DCR1的氨基酸序列(含信号肽)(SEQ ID NO:7)
8. 137DCR2的氨基酸序列(含信号肽)(SEQ ID NO:8)
9. 137DCR3的氨基酸序列(含信号肽)(SEQ ID NO:9)
10. 137DCR4的氨基酸序列(含信号肽)(SEQ ID NO:10)
11. 137DCR1的氨基酸序列(不含信号肽)(SEQ ID NO:11)
12. 137DCR2的氨基酸序列(不含信号肽)(SEQ ID NO:12)
13. 137DCR3的氨基酸序列(不含信号肽)(SEQ ID NO:13)
14. 137DCR4的氨基酸序列(不含信号肽)(SEQ ID NO:14)
15. 137DCR1的核酸序列(含信号肽)(SEQ ID NO:15)
16. 137DCR2的核酸序列(含信号肽)(SEQ ID NO:16)
17. 137DCR3的核酸序列(含信号肽)(SEQ ID NO:17)
18. 137DCR4的核酸序列(含信号肽)(SEQ ID NO:18)
19. 137DCR1的核酸序列(不含信号肽)(SEQ ID NO:19)
20. 137DCR2的核酸序列(不含信号肽)(SEQ ID NO:20)
21. 137DCR3的核酸序列(不含信号肽)(SEQ ID NO:21)
22. 137DCR4的核酸序列(不含信号肽)(SEQ ID NO:22)
23.meso G1 CAR的氨基酸序列(SEQ ID NO:23)
24.meso G1 CAR的核酸序列(SEQ ID NO:24)
25.CD3ζ的酪氨酸活化基序(SEQ ID NO:25)
26.meso G2 CAR的核苷酸序列(SEQ ID NO:26)
27.PS328b载体的核酸序列(SEQ ID NO:27)
28.引物CD137-F的碱基序列(SEQ ID NO:28)
29.引物CD137-R的碱基序列(SEQ ID NO:29)
30.探针Taqman中的碱基序列(SEQ ID NO:30)
31.CD28胞外激活型单链抗体的氨基酸序列(SEQ ID NO:31)
32. 28DCR1的氨基酸序列(含信号肽)(SEQ ID NO:32)
33. 28DCR2的氨基酸序列(含信号肽)(SEQ ID NO:33)
34. 28DCR3的氨基酸序列(含信号肽)(SEQ ID NO:34)
35. 28DCR4的氨基酸序列(含信号肽)(SEQ ID NO:35)
36. 28DCR1的氨基酸序列(不含信号肽)(SEQ ID NO:36)
37. 28DCR2的氨基酸序列(不含信号肽)(SEQ ID NO:37)
38. 28DCR3的氨基酸序列(不含信号肽)(SEQ ID NO:38)
39. 28DCR4的氨基酸序列(不含信号肽)(SEQ ID NO:39)
40. 28DCR1的核酸序列(含信号肽)(SEQ ID NO:40)
41. 28DCR2的核酸序列(含信号肽)(SEQ ID NO:41)
42. 28DCR3的核酸序列(含信号肽)(SEQ ID NO:42)
43. 28DCR4的核酸序列(含信号肽)(SEQ ID NO:43)
44. 28DCR1的核酸序列(不含信号肽)(SEQ ID NO:44)
45. 28DCR2的核酸序列(不含信号肽)(SEQ ID NO:45)
46. 28DCR3的核酸序列(不含信号肽)(SEQ ID NO:46)
47. 28DCR4的核酸序列(不含信号肽)(SEQ ID NO:47)
48.Muc1 G1 CAR的氨基酸序列(SEQ ID NO:48)
49.Muc1 G1 CAR的核酸序列(SEQ ID NO:49)
50.Muc1 G2 CAR的核苷酸序列(SEQ ID NO:50)
51.引物CD28-F的碱基序列(SEQ ID NO:51)
52.引物CD28-R的碱基序列(SEQ ID NO:52)
53.探针Taqman中的碱基序列(SEQ ID NO:53)
54.CD8信号肽的氨基酸序列(SEQ ID NO:54)
55.CD40胞外激活型单链抗体的氨基酸序列(SEQ ID NO:55)
56.IgG4Fc CH2CH3铰链区的氨基酸序列(SEQ ID NO:56)
57. 40DCR1的氨基酸序列(含信号肽)(SEQ ID NO:57)
58. 40DCR2的氨基酸序列(含信号肽)(SEQ ID NO:58)
59. 40DCR3的氨基酸序列(含信号肽)(SEQ ID NO:59)
60. 40DCR4的氨基酸序列(含信号肽)(SEQ ID NO:60)
61. 40DCR1的氨基酸序列(不含信号肽)(SEQ ID NO:61)
62. 40DCR2的氨基酸序列(不含信号肽)(SEQ ID NO:62)
63. 40DCR3的氨基酸序列(不含信号肽)(SEQ ID NO:63)
64. 40DCR4的氨基酸序列(不含信号肽)(SEQ ID NO:64)
65. 40DCR1的核酸序列(含信号肽)(SEQ ID NO:65)
66. 40DCR2的核酸序列(含信号肽)(SEQ ID NO:66)
67. 40DCR3的核酸序列(含信号肽)(SEQ ID NO:67)
68. 40DCR4的核酸序列(含信号肽)(SEQ ID NO:68)
69. 40DCR1的核酸序列(不含信号肽)(SEQ ID NO:69)
70. 28DCR2的核酸序列(不含信号肽)(SEQ ID NO:70)
71. 40DCR3的核酸序列(不含信号肽)(SEQ ID NO:71)
72. 40DCR4的核酸序列(不含信号肽)(SEQ ID NO:72)
73.EGFR G1 CAR的氨基酸序列(SEQ ID NO:73)
74.EGFR G1 CAR的核酸序列(SEQ ID NO:74)
75.EGFR G2 CAR的核苷酸序列(SEQ ID NO:75)
76.引物CD40-F的碱基序列(SEQ ID NO:76)
77.引物CD40-R的碱基序列(SEQ ID NO:77)
78.探针Taqman中的碱基序列(SEQ ID NO:78)
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
实施例1-(1)：5个重组质粒即pNB328-meso CAR G1、pNB328-meso G2 CAR、
PS328b 137DCR1、PS328b 137DCR2和PS328b 137DCR3的构建
1.人工合成137DCR1的基因(SEQ ID NO:15)、137DCR2的基因(SEQ ID NO:16)、137DCR3的基因(SEQ ID NO:17)、meso G1 CAR基因(SEQ ID NO:24)以及meso G2 CAR基因(SEQ ID NO:26)，其结构示意图分别如图1A-1、图1B-1、图1C-1、图1E-1和图1F-1所示。将合成的5个基因分别装入PNB328载体和PS328b载体，EcoRI和SalI酶切位点之间，
pNB328载体中包含EF1α启动子、PB转座子等原件，pNB328载体的构建请参见WO2017054647A1的实施例2。PS328b是人工合成序列，由上海捷瑞生物工程有限公司合成，序列如SEQ ID NO:27所示。
构建出的重组质粒分别命名为pNB328-meso G1 CAR质粒、pNB328-meso G2 CAR质粒、PS328b 137DCR1质粒、PS328b 137DCR2质粒和PS328b 137DCR3质粒。构建出的重组质粒分别可以携带外源基因整合到宿主细胞的基因组中。
实施例1-(2)：5个重组质粒即重组质粒pNB328-Muc1 G1 CAR、pNB328-Muc1 G2
CAR、PS328b 28DCR1、PS328b 28DCR2和PS328b 28DCR3的构建
1.人工合成28DCR1的基因(SEQ ID NO:40)、28DCR2的基因(SEQ ID NO:41)、28DCR3的基因(SEQ ID NO:42)、Muc1 G1 CAR基因(SEQ ID NO:49)以及Muc1 G2 CAR基因(SEQ ID NO:50)，其结构示意图分别如图1A-2、图1B-2、图1C-2、图1E-2和图1F-2所示。将合成的5个基因分别装入PNB328载体和PS328b载体，EcoRI和SalI酶切位点之间，
pNB328载体中包含EF1α启动子、PB转座子等原件，pNB328载体的构建请参 见WO2017054647A1的实施例2。PS328b是人工合成序列，由上海捷瑞生物工程有限公司合成，序列如SEQ ID NO:27所示。
构建出的重组质粒分别命名为pNB328-Muc1 G1 CAR质粒、pNB328-Muc1 G2 CAR质粒、PS328b 28DCR1质粒、PS328b 28DCR2质粒和PS328b 28DCR3质粒。构建出的重组质粒分别可以携带外源基因整合到宿主细胞的基因组中。
实施例1-(3)：5种重组质粒即pNB328-EGFR G1 CAR、pNB328-EGFR G2 CAR、
PS328b 40DCR1、PS328b 40DCR2和PS328b 40DCR3的构建
1.人工合成40DCR1的基因(SEQ ID NO:65)、40DCR2的基因(SEQ ID NO:66)、40DCR3的基因(SEQ ID NO:67)、EGFR G1 CAR基因(SEQ ID NO:74)以及EGFR G2 CAR基因(SEQ ID NO:75)，其结构示意图分别如图1A-3、图1B-3、图1C-3、图1F-3和图1D-3所示。将合成的5个基因分别装入pNB328载体和PS328b载体，EcoRI和SalI酶切位点之间，
pNB328载体中包含EF1α启动子、PB转座子等原件，pNB328载体的构建请参见WO2017054647A1的实施例2。PS328b是人工合成序列，由上海捷瑞生物工程有限公司合成，序列如SEQ ID NO:27所示。
构建出的重组质粒分别命名为pNB328-EGFR G1 CAR质粒、pNB328-EGFR G2 CAR质粒、PS328b 40DCR1质粒、PS328b 40DCR2质粒和PS328b 40DCR3质粒。构建出的重组质粒分别可以携带外源基因整合到宿主细胞的基因组中。
实施例2-(1)：9种嵌合型抗原受体修饰T细胞即重组细胞meso G1 CAR、meso G2
CAR、meso G1 CAR-137DCR1、meso G1 CAR-137DCR2、meso G1 CAR-137DCR3、
137DCR1、137DCR2、137DCR3和Mock T的构建和鉴定
(1)9种重组细胞的构建
将外周血单核细胞(PBMCs)贴壁培养2-4h，其中未贴壁的悬浮细胞即为初始T细胞。将悬浮细胞收集到15ml离心管中，1200rmp离心3min，弃上清；加入生理盐水，1200rmp离心3min，弃生理盐水，并重复“加入生理盐水，1200rmp离心3min，弃生理盐水”的步骤3次。
取8个1.5ml离心管，每管加入5×10
6个上述细胞，编号分别为a、b、c、d、e、f、g、h，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，每管按比例 加入电转试剂共100μl，其中：
a管加入pNB328-meso G1 CAR质粒8μg，
b管加入pNB328-meso G2 CAR质粒8μg，
c管加入PS328b 137DCR1质粒和pNB328-meso G1 CAR质粒各4μg，
d管加入PS328b 137DCR2质粒和pNB328-meso G1 CAR质粒各4μg，
e管加入PS328b 137DCR3质粒和pNB328-meso G1 CAR质粒各4μg，
f管加入PS328b 137DCR1质粒8μg，
g管加入PS328b 137DCR2质粒8μg，
h管加入PS328b 137DCR3质粒8μg，
将上述8管各自重悬混匀，分别得到混合液。
将各混合液分别转移至电转杯中，放入电转仪，选取所需程序，进行电击；使用试剂盒中的微量吸管将电转好的细胞悬液转移到加好培液的六孔板中(含2％FBS的AIM-Ⅴ培液)，混匀，置于37℃，5％CO
2培养箱培养6小时，然后加入刺激因子IL-2和meso/anti-CD28，37℃，5％CO
2培养3-4天，观察T细胞的生长情况。由此分别获得了表达pNB328-meso G1 CAR、pNB328-meso G2 CAR、pNB328-meso G1 CAR-137DCR1、pNB328-meso G1 CAR-137DCR2、pNB328-meso G1 CAR-137DCR3、PS328b 137DCR1、PS328b 137DCR2和PS328b 137DCR3基因的重组T细胞，依次命名为重组细胞meso G1 CAR、重组细胞meso G2 CAR、重组细胞meso G1 CAR-137DCR1、重组细胞meso G1 CAR-137DCR2和重组细胞meso G1 CAR-137DCR3、重组细胞137DCR1、重组细胞137DCR2以及重组细胞137DCR3。
此外，在另一离心管(编号i)，加入5×10
6个细胞，1200rmp离心3min，弃上清，取电转试剂盒(购自Lonza公司)，按比例加入电转试剂共100μl，并加入8μg对照质粒(PNB328)，按前文所述方法构建得到对照T细胞，命名为Mock T。
(2)阳性重组细胞的鉴定
①western blot方法检测meso G1 CAR或meso G2 CAR基因的表达
分别收集上述重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR1、meso G1CAR-137DCR2、meso G1 CAR-137DCR3和Mock-T细胞，生理盐水洗涤两遍，
分别加入160μl细胞裂解液，冰上放置10min；待细胞充分裂解后，12000rmp，4℃，离心10min，收集上清。加入40μl 5×loading Buffer，100℃煮10min，然后冰上放置5min。
使用CD3ζ抗体(购自abcam公司)、GAPDH抗体(购自Beyotime公司)、HRP 羊抗鼠二抗(购自Jackson公司)，通过western blot等等检测前面构建的重组细胞中的CD3ζ的表达，结果如图2A-1所示。
结果显示，CD3ζ在这构建的重组T细胞中均为高表达。
②RT-PCR检测137DCR1-3基因表达
分别提取重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR1、meso G1 CAR-137DCR2、meso G1 CAR-137DCR3和Mock-T的基因组DNA(试剂盒法)，实验步骤参照试剂盒内附带的说明书。
测定各重组细胞DNA的浓度，采用荧光实时定量PCR的方法检测137DCR基因的表达水平，反应为：95℃，15s；95℃，5s；60℃，15s。40个循环。
PCR反应体系(20μl)如下：
Taqman：10μl
CD137-F:0.4μl
CD137-R:0.4μl
Cd137-probe:0.2μl
Actin mix:1μl
H
2O:7μl
引物序列如下：
CD137-F：CGAGTCACCATATCAGTA(SEQ ID NO:28)
CD137-R：CGAAGTACCAGTCATAATTC(SEQ ID NO:29)
Taqman：5'FAM-CCTCGCACAGTAATATACAGCCGT-Trama(SEQ ID NO:30)
结果如图2B-1所示。结果显示，重组细胞meso G1 CAR-137DCR1、meso G1 CAR-137DCR2、meso G1 CAR-137DCR3细胞中，137DCR1、137DCR2或137DCR3基因的表达量很高。
实施例2-(2)：9种嵌合型抗原受体修饰T细胞即重组细胞Muc1 G1 CAR、Muc1
G2 CAR、Muc1 G1 CAR-28DCR1、Muc1 G1 CAR-28DCR2、Muc1 G1 CAR-28DCR3、
28DCR1、28DCR2、28DCR3和Mock T的构建和鉴定
(1)9种重组细胞的构建
将外周血单核细胞(PBMCs)贴壁培养2-4h，其中未贴壁的悬浮细胞即为初始T细胞。将悬浮细胞收集到15ml离心管中，1200rmp离心3min，弃上清；加入生理盐水， 1200rmp离心3min，弃生理盐水，并重复“加入生理盐水，1200rmp离心3min，弃生理盐水”的步骤3次。
取8个1.5ml离心管，每管加入5×106个上述细胞，编号分别为a、b、c、d、e、f、g、h，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，每管按比例加入电转试剂共100μl，其中：
a管加入pNB328-Muc1 G1 CAR质粒8μg，
b管加入pNB328-Muc1 G2 CAR质粒8μg，
c管加入PS328b 28DCR1质粒和pNB328-Muc1 G1 CAR质粒各4μg，
d管加入PS328b 28DCR2质粒和pNB328-Muc1 G1 CAR质粒各4μg，
e管加入PS328b 28DCR3质粒和pNB328-Muc1 G1 CAR质粒各4μg，
f管加入PS328b 28DCR1质粒8μg，
g管加入PS328b 28DCR2质粒8μg，
h管加入PS328b 28DCR3质粒8μg，
将上述8管各自重悬混匀，分别得到混合液。
将各混合液分别转移至电转杯中，放入电转仪，选取所需程序，进行电击；使用试剂盒中的微量吸管将电转好的细胞悬液转移到加好培液的六孔板中(含2％FBS的AIM-Ⅴ培液)，混匀，置于37℃，5％CO
2培养箱培养6小时，然后加入刺激因子IL-2和Muc1/anti-CD28，37℃，5％CO
2培养3-4天，观察T细胞的生长情况。由此分别获得了表达pNB328-Muc1 G1 CAR、pNB328-Muc1 G2 CAR、pNB328-Muc1 G1 CAR-28DCR1、pNB328-Muc1 G1 CAR-28DCR2、pNB328-Muc1 G1 CAR-28DCR3、PS328b 28DCR1、PS328b 28DCR2和PS328b 28DCR3基因的重组T细胞，依次命名为重组细胞Muc1 G1 CAR、重组细胞Muc1 G2 CAR、重组细胞Muc1 G1 CAR-28DCR1、重组细胞Muc1 G1 CAR-28DCR2和重组细胞Muc1 G1 CAR-28DCR3、重组细胞28DCR1、重组细胞28DCR2以及重组细胞28DCR3。
此外，在另一离心管(编号i)，加入5×10
6个细胞，1200rmp离心3min，弃上清，取电转试剂盒(购自Lonza公司)，按比例加入电转试剂共100μl，并加入8μg对照质粒(PNB328)，按前文所述方法构建得到对照T细胞，命名为Mock T。
(2)阳性重组细胞的鉴定
①western blot方法检测Muc1 G1 CAR或Muc1 G2 CAR基因的表达
分别收集上述重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR1、 Muc1 G1CAR-28DCR2、Muc1 G1 CAR-28DCR3和Mock-T细胞，生理盐水洗涤两遍，
分别加入160μl细胞裂解液，冰上放置10min；待细胞充分裂解后，12000rmp，4℃，离心10min，收集上清。加入40μl 5×loading Buffer，100℃煮10min，然后冰上放置5min。
使用CD3ζ抗体(购自abcam公司)、GAPDH抗体(购自Beyotime公司)、HRP羊抗鼠二抗(购自Jackson公司)，通过western blot等等检测前面构建的重组细胞中的CD3ζ的表达，结果如图2A-2所示。
结果显示，CD3ζ在这构建的重组T细胞中均为高表达。
②RT-PCR检测28DCR1-3基因表达
分别提取重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR1、Muc1 G1 CAR-28DCR2、Muc1 G1 CAR-28DCR3和Mock-T的基因组DNA(试剂盒法)，实验步骤参照试剂盒内附带的说明书。
测定各重组细胞DNA的浓度，采用荧光实时定量PCR的方法检测28DCR基因的表达水平，反应为：95℃，15s；95℃，5s；60℃，15s。40个循环。
PCR反应体系(20μl)如下：
Taqman：10μl
CD28-F:0.4μl
CD28-R:0.4μl
CD28-probe:0.2μl
Actin mix:1μl
H
2O:7μl
引物序列如下：
CD28-F：GCTTCTGGATACACCTTC(SEQ ID NO:51)
CD28-R：CCTTGAACTTCTCATTATAGTTAG(SEQ ID NO:52)
Taqman：5'FAM-AATACATCCAATCCACTCAAGCC-Trama(SEQ ID NO:53)
结果如图2B-2所示。结果显示，重组细胞Muc1 G1 CAR-28DCR1、Muc1 G1 CAR-28DCR2、Muc1 G1 CAR-28DCR3细胞中，28DCR1、28DCR2或28DCR3基因的表达量很高。
实施例2-(3)：9种嵌合型抗原受体修饰T细胞即重组细胞EGFR G1 CAR、EGFR
G2 CAR、EGFR G1 CAR-40DCR1、EGFR G1 CAR-40DCR2、EGFR G1
CAR-40DCR3、40DCR1、40DCR2、40DCR3和Mock T的构建和鉴定
(1)9种重组细胞的构建
将外周血单核细胞(PBMCs)贴壁培养2-4h，其中未贴壁的悬浮细胞即为初始T细胞。将悬浮细胞收集到15ml离心管中，1200rmp离心3min，弃上清；加入生理盐水，1200rmp离心3min，弃生理盐水，并重复“加入生理盐水，1200rmp离心3min，弃生理盐水”的步骤3次。
取8个1.5ml离心管，每管加入5×106个上述细胞，编号分别为a、b、c、d、e、f、g、h，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，每管按比例加入电转试剂共100μl，其中：
a管加入pNB328-EGFR G1 CAR质粒8μg，
b管加入pNB328-EGFR G2 CAR质粒8μg，
c管加入PS328b 40DCR1质粒和pNB328-EGFR G1 CAR质粒各4μg，
d管加入PS328b 40DCR2质粒和pNB328-EGFR G1 CAR质粒各4μg，
e管加入PS328b 40DCR3质粒和pNB328-EGFR G1 CAR质粒各4μg，
f管加入PS328b 40DCR1质粒8μg，
g管加入PS328b 40DCR2质粒8μg，
h管加入PS328b 40DCR3质粒8μg，
将上述8管各自重悬混匀，分别得到混合液。
将各混合液分别转移至电转杯中，放入电转仪，选取所需程序，进行电击；使用试剂盒中的微量吸管将电转好的细胞悬液转移到加好培液的六孔板中(含2％FBS的AIM-Ⅴ培液)，混匀，置于37℃，5％CO
2培养箱培养6小时，然后加入刺激因子IL-2和EGFR/anti-CD28，37℃，5％CO
2培养3-4天，观察T细胞的生长情况。由此分别获得了表达pNB328-EGFR G1 CAR、pNB328-EGFR G2 CAR、pNB328-EGFR G1 CAR-40DCR1、pNB328-EGFR G1 CAR-40DCR2、pNB328-EGFR G1 CAR-40DCR3、PS328b 40DCR1、PS328b 40DCR2和PS328b 40DCR3基因的重组T细胞，依次命名为重组细胞EGFR G1 CAR、重组细胞EGFR G2 CAR、重组细胞EGFR G1 CAR-40DCR1、重组细胞EGFR G1 CAR-40DCR2和重组细胞EGFR G1 CAR-40DCR3、重组细胞40DCR1、重组细胞40DCR2以及重组细胞40DCR3。
此外，在另一离心管(编号j)，加入5×10
6个细胞，1200rmp离心3min，弃上清，取电转试剂盒(购自Lonza公司)，按比例加入电转试剂共100μl，并加入8μg对照质粒 (PNB328)，按前文所述方法构建得到对照T细胞，命名为Mock T。
(2)阳性重组细胞的鉴定
①western blot方法检测EGFR G1 CAR或EGFR G2 CAR基因的表达
分别收集上述重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR1、EGFR G1CAR-40DCR2、EGFR G1 CAR-40DCR3和Mock-T细胞，生理盐水洗涤两遍，
分别加入160μl细胞裂解液，冰上放置10min；待细胞充分裂解后，12000rmp，4℃，离心10min，收集上清。加入40μl 5×loading Buffer，100℃煮10min，然后冰上放置5min。
使用CD3ζ抗体(购自abcam公司)、GAPDH抗体(购自Beyotime公司)、HRP羊抗鼠二抗(购自Jackson公司)，通过western blot等等检测前面构建的5种重组细胞中的CD3ζ的表达，结果如图2A-3所示。
结果显示，CD3ζ在这构建的5种重组T细胞中均为高表达。
②RT-PCR检测40DCR基因表达
分别提取重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR1、EGFR G1 CAR-40DCR2、EGFR G1 CAR-40DCR3和Mock-T的基因组DNA(试剂盒法)，实验步骤参照试剂盒内附带的说明书。
测定各重组细胞DNA的浓度，采用荧光实时定量PCR的方法检测40DCR基因的表达水平，反应为：95℃，15s；95℃，5s；60℃，15s。40个循环。
PCR反应体系(20μl)如下：
Taqman：10μl
CD40-F:0.4μl
CD40-R:0.4μl
CD40-probe:0.2μl
Actin mix:1μl
H
2O:7μl
引物序列如下：
CD40-F：ACCTCCTGATCTATACTG(SEQ ID NO:76)
CD40-R：GATGGTGAGAGTGAAATC(SEQ ID NO:77)
Taqman：5'FAM-CACTGCCGCTGAACCTTGATG-Trama(SEQ ID NO:78)
结果如图2B-3所示。结果显示，重组细胞EGFR G1 CAR-40DCR1、EGFR G1 CAR-40DCR2、EGFR G1 CAR-40DCR3细胞中，40DCR基因的表达量很高。
实施例3-(1)：流式检测细胞技术检测细胞增殖活力
1.实验样品、试剂和仪器
实施例2-(1)制得的重组细胞137DCR1、137DCR2、137DCR 3和Mock T。
在50ml离心管中配制含有2％FBS的PBS(1ml Hyclone FBS+49ml PBS)；用ddH
2O将10×的BD Perm/Wash
TM buffer稀释10倍，置于冰上；Hoechst 33342原液用ddH
2O以1:100比例稀释成工作液(1μl/test)。
低温离心机(4℃预冷)。
2.实验方法
实验过程中所有的试剂均置于冰上。
具体步骤如下：
(1)分别收取1×10
6-2×10
6的上述各细胞于1.5ml离心管中，加入适量含有2％FBS的PBS，5000rpm，4℃离心5min，弃上清；
(2)用100μl Fixation/Permeabilization Solution重悬细胞，重悬要充分，动作要轻柔，4℃固定透化20min；
(3)加入1ml工作浓度的Perm/Wash
TM buffer，混匀，7000rpm，4℃离心5min，弃上清，重复清洗一次；
(4)用100μl工作浓度的Perm/Wash
TM buffer重悬细胞。加入2-3μl Ki-67-APC抗体，4℃避光孵育30min；
(5)加入1ml工作浓度的Perm/Wash
TM buffer，混匀，7000rpm，4℃离心5min，弃上清，重复清洗一次；
(6)用100μl工作浓度的Perm/Wash
TM buffer重悬细胞。加入1μl工作浓度的Hoechst 33342，冰上孵育15min；
(7)补加400μl工作浓度的Perm/Wash
TM buffer，混匀，上机检测。
3.实验结果
结果如图3A-1至3D-1所示。
结果显示，Mock T增殖速率最慢，137DCR1增殖速率较慢，137DCR2增殖速率较快，137DCR3增殖速率最快。
实施例4-(1)：细胞增殖活力试剂盒检测细胞技术检测细胞增殖活力
1.实验样品和试剂
实施例2-(1)制得的重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR2和Mock T。
2.实验方法
(1)准备96孔白色板，分别取培养第8天的上述各细胞，每孔加入100μL含细胞的AIM-V培养基。
(2)准备不含细胞的空白对照，以获得背景荧光值。
(3)在孔板中加入待测复合物，在培养箱中孵育30min。
(4)加入100μL CellTiter-Glo试剂，在震荡仪上混匀2min，并在室温下孵育10min，读数。
(5)培养的第8、9、10天，每天都按以上步骤检测。
3.实验结果
如图4-1所示。
结果表明，Mock T增殖速率最慢，meso G1 CAR增殖速率较慢，meso G2 CAR增殖速率较快，meso G1 CAR-137DCR2增殖速率最快。
实施例4-(2)：细胞增殖活力试剂盒检测细胞技术检测细胞增殖活力
1.实验样品和试剂
实施例2-(2)制得的重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR2、28DCR1、28DCR2、28DCR3和Mock T。
2.实验方法
(1)准备96孔白色板，分别取培养第8天的上述各细胞，每孔加入100μL含细胞的AIM-V培养基。
(2)准备不含细胞的空白对照，以获得背景荧光值。
(3)在孔板中加入待测复合物，在培养箱中孵育30min。
(4)加入100μL CellTiter-Glo试剂，在震荡仪上混匀2min，并在室温下孵育10min，读数。
(5)培养的第8、9、10天，每天都按以上步骤检测。
3.实验结果
分别如图3-2和图4-2所示。
图3-2表明，Mock T增殖速率最慢，28DCR增殖速率均较快。
图4-2表明，Mock T增殖速率最慢，Muc1 G1 CAR增殖速率较慢，Muc1 G2 CAR增殖速率较快，Muc1 G1 CAR-28DCR2增殖速率最快。
实施例4-(3)：细胞增殖活力试剂盒检测细胞技术检测细胞增殖活力
1.实验样品和试剂
实施例2-(3)制得的重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR2、40DCR1、40DCR2、40DCR3和Mock T。
2.实验方法
(1)准备96孔白色板，分别取培养第8天的上述各细胞，每孔加入100μL含细胞的AIM-V培养基。
(2)准备不含细胞的空白对照，以获得背景荧光值。
(3)在孔板中加入待测复合物，在培养箱中孵育30min。
(4)加入100μL CellTiter-Glo试剂，在震荡仪上混匀2min，并在室温下孵育10min，读数。
(5)培养的第8、9、10天，每天都按以上步骤检测。
3.实验结果
分别如图3-3和图4-3所示。
图3-3表明，Mock T增殖速率最慢，40DCR增殖速率均较快。
图4-3表明，Mock T增殖速率最慢，EGFR G1 CAR增殖速率较慢，EGFR G2 CAR增殖速率较快，EGFR G1 CAR-40DCR2增殖速率最快。
实施例5-(1)：流式检测间皮素抗原刺激下双向共刺激分子激活受体137DCR联合
meso G1 CAR-T细胞表型
1.实验样品
实施例2-(1)制得的重组细胞137DCR1，137DCR2，137DCR 3，meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR3和Mock T。
2.实验方法
分别收集上述各细胞，计数后以1×10
6个细胞/管分别加入1.5ml的EP管中，PBS洗两遍，1200rpm离心5min，分别加入2μl的同型对照抗体IgG1-PE，荧光流式抗体anti-CD137、同型对照(IgG1FITC+IgG1PC5+IgG1PE)，(anti-CD45RO-PC5，anti-CD62L-FITC,anti-CCR7-PE)，轻弹沉淀使其混合均匀，室温避光孵育30min，PBS清洗一遍，加400μlPBS将细胞转移至流式管中，上机检测。
3.实验结果
如图5A-1至5D-1、图6A-1至6D-1、图7A-1至7D-1和图8A-1至8D-1所示。其中：
图5A-1至5D-1为3种137DCR1、137DCR2、137DCR3的3种单转细胞，CD137表型相对于Mock T有很大提高。
图6A-1至6D-1为Mock T、meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR2的CD137表型，相对于其他三组，meso G1 CAR-137DCR2有很大提高。
图7A-1至7D-1为Mock T、meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR2的CD45RO表型，表示细胞活化程度，均已大量活化。
图8A-1至8D-1为Mock T、meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR2的记忆T表型,相对于其他三组，meso G1 CAR-137DCR2能促进记忆T的形成。
实施例5-(2)：流式检测Muc1抗原刺激下双向共刺激分子激活受体28DCR联合
Muc1 G1 CAR-T细胞表型
1.实验样品
实施例2-(2)制得的重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR3、28DCR1、28DCR2、28DCR 3和Mock T。
2.实验方法
分别收集上述各细胞，计数后以1×10
6个细胞/管分别加入1.5ml的EP管中，PBS洗两遍，1200rpm离心5min，分别加入2μl的同型对照抗体IgG1-PE，荧光流式抗体anti-CD137、同型对照(IgG1FITC+IgG1PC5+IgG1PE)，(anti-CD45RO-PC5，anti-CD62L-FITC,anti-CCR7-PE)，轻弹沉淀使其混合均匀，室温避光孵育30min， PBS清洗一遍，加400μlPBS将细胞转移至流式管中，上机检测。
3.实验结果
如图5A-2至5D-2、图6A-2至6D-2、图7A-2至7D-2和图8A-2至8D-2所示。其中：
图5A-2至5D-2为3种28DCR1、28DCR2、28DCR3的3种单转细胞，CD137表型相对于Mock T有很大提高。
图6A-2至6D-2为Mock T、Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR2的CD137表型，相对于其他三组，Muc1 G1 CAR-28DCR2有很大提高。
图7A-2至7D-2为Mock T、Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR2的CD45RO表型，表示细胞活化程度，均已大量活化。
图8A-2至8D-2为Mock T、Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR2的记忆T表型,相对于其他三组，Muc1 G1 CAR-28DCR2能促进记忆T的形成。
实施例5-(3)：流式检测EGFR抗原刺激下双向共刺激分子激活受体40DCR联合
EGFR G1 CAR-T细胞表型
1.实验样品
实施例2-(3)制得的重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR3、40DCR1，40DCR2，40DCR 3和Mock T。
2.实验方法
分别收集上述各细胞，计数后以1×10
6个细胞/管分别加入1.5ml的EP管中，PBS洗两遍，1200rpm离心5min，分别加入2μl的同型对照抗体IgG1-PE，荧光流式抗体anti-CD137、同型对照(IgG1FITC+IgG1PC5+IgG1PE)，(anti-CD45RO-PC5，anti-CD62L-FITC,anti-CCR7-PE)，轻弹沉淀使其混合均匀，室温避光孵育30min，PBS清洗一遍，加400μlPBS将细胞转移至流式管中，上机检测。
3.实验结果
如图5A-3至5D-3、图6A-3至6D-3、图7A-3至7D-3和图8A-3至8D-3所示。其中：
图5A-3至5D-3为3种40DCR1、40DCR2、40DCR3的3种单转细胞，CD137表型相对于Mock T有很大提高；
图6A-3至6D-3为Mock T、EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR2的CD137表型，相对于其他三组，EGFR G1 CAR-40DCR2有很大提高；
图7A-3至7D-3为Mock T、EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR2的CD45RO表型，表示细胞活化程度，均已大量活化；
图8A-3至8D-3为Mock T、EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR2的记忆T表型,相对于其他三组，EGFR G1 CAR-40DCR2能促进记忆T的形成。
实施例6-(1)：实时无标记细胞功能分析仪检测双向共刺激分子激活受体137DCR
联合meso G1 CAR-T细胞对肿瘤细胞的体外杀伤作用
1.实验样品
效应细胞：实施例2-(1)制得的重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR1、meso G1 CAR-137DCR3和Mock T。
靶细胞：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3(均购买于美国菌种保藏中心ATCC)。
2.实验方法
选取MHC class I分型匹配的效应细胞与靶细胞，应用实时无标记细胞功能分析仪(RTCA)检测上述细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μl DMEM或1640培养液，放入仪器中，选择step 1，调零；
(2)靶细胞铺板：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3分别按每孔10
4个细胞/50μl铺在含有检测电极的板中，放置数分钟，待细胞稳定一下，再放入仪器中，开始step 2，培养细胞；
(3)加入效应细胞：靶细胞培养24h后，暂停step 2，加入效应细胞，每孔50μl，效靶比分别设置为8:1、4:(肿瘤细胞均为10
4个)1，以未转质粒的Mock T细胞作为对照，开始step 3，继续共培养24h后，观察细胞增殖曲线。
2.实验结果
如图9A-1至9D-1所示。
结果显示，Mock T对肿瘤细胞的杀伤作用最弱，meso G1 CAR对肿瘤细胞的杀伤作用较弱，meso G2 CAR对肿瘤细胞的杀伤作用较强，meso G1 CAR-137DCR1、 meso G1 CAR-137DCR3对肿瘤细胞的杀伤作用最强。
实施例6-(2)：实时无标记细胞功能分析仪检测双向共刺激分子激活受体28DCR
联合Muc1 G1 CAR-T细胞对肿瘤细胞的体外杀伤作用
1.实验样品
效应细胞：实施例2-(2)制得的重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR1、Muc1 G1 CAR-28DCR3、28DCR1、28DCR2以及28DCR3和Mock T。
靶细胞：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3(均购买于美国菌种保藏中心ATCC)。
2.实验方法
选取MHC class I分型匹配的效应细胞与靶细胞，应用实时无标记细胞功能分析仪(RTCA)检测上述细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μl DMEM或1640培养液，放入仪器中，选择step 1，调零；
(2)靶细胞铺板：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3分别按每孔10
4个细胞/50μl铺在含有检测电极的板中，放置数分钟，待细胞稳定一下，再放入仪器中，开始step 2，培养细胞；
(3)加入效应细胞：靶细胞培养24h后，暂停step 2，加入效应细胞，每孔50μl，效靶比分别设置为8:1、4:(肿瘤细胞均为10
4个)1，以未转质粒的Mock T细胞作为对照，开始step 3，继续共培养24h后，观察细胞增殖曲线。
2.实验结果
如图9A-2至9D-2所示。
结果显示，Mock T对肿瘤细胞的杀伤作用最弱，Muc1 G1 CAR对肿瘤细胞的杀伤作用较弱，Muc1 G2 CAR对肿瘤细胞的杀伤作用较强，Muc1 G1 CAR-28DCR1、Muc1 G1 CAR-28DCR3对肿瘤细胞的杀伤作用最强。
实施例6-(3)：实时无标记细胞功能分析仪检测双向共刺激分子激活受体40DCR
联合EGFR G1 CAR-T细胞对肿瘤细胞的体外杀伤作用
1.实验样品
效应细胞：实施例2-(3)制得的重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR1、EGFR G1 CAR-40DCR3、40DCR1、40DCR2、40DCR3和Mock T。
靶细胞：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3(均购买于美国菌种保藏中心ATCC)。
2.实验方法
选取MHC class I分型匹配的效应细胞与靶细胞，应用实时无标记细胞功能分析仪(RTCA)检测上述细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μl DMEM或1640培养液，放入仪器中，选择step 1，调零；
(2)靶细胞铺板：宫颈癌细胞Hela、卵巢癌细胞SK-OV-3分别按每孔10
4个细胞/50μl铺在含有检测电极的板中，放置数分钟，待细胞稳定一下，再放入仪器中，开始step 2，培养细胞；
(3)加入效应细胞：靶细胞培养24h后，暂停step 2，加入效应细胞，每孔50μl，效靶比分别设置为8:1、4:(肿瘤细胞均为10
4个)1，以未转质粒的Mock T细胞作为对照，开始step 3，继续共培养24h后，观察细胞增殖曲线。
2.实验结果
如图9A-3至9D-3所示。
结果显示，Mock T对肿瘤细胞的杀伤作用最弱，EGFR G1 CAR对肿瘤细胞的杀伤作用较弱，EGFR G2 CAR对肿瘤细胞的杀伤作用较强，EGFR G1 CAR-40DCR1、EGFR G1 CAR-40DCR3对肿瘤细胞的杀伤作用最强。
实施例7-(1)：流式检测间皮素抗原刺激下meso CAR-T细胞因子分泌量
1.实验样品
实施例2-(1)制得的重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR1和Mock T。
2.实验方法
1.用5μg/ml的间皮素抗原包被96孔板，4℃包被过夜，PBS清洗3遍，分别加入1×10
5的各样品细胞，培养24h后收集细胞上清。用BD
TMCBA Human Th1/Th2Cytokine Kit II检测meso CAR-T细胞受间皮素抗原刺激后细胞因子的分泌情况。
具体步骤如下：
(1)混合人的IL-2、IL-4、IL-6、IL-10、TNF、IFN-γ捕获磁珠，涡旋振荡混匀捕获磁珠，每管加入50μl混匀后的捕获磁珠；
(2)加入50μl人的Th1/Th2细胞因子标准品(倍比稀释5000pg/ml、2500pg/ml、 1250pg/ml、625pg/ml、312.5pg/ml、156pg/ml、80pg/ml、40pg/ml、20pg/ml、0pg/ml)和50μl的待测样品(经稀释液2倍稀释)。
(3)每管加入50μl的人的Th1/Th2-II-PE的检测抗体；
(4)室温避光孵育3h；
(5)每管加入1ml的Wash Buffer，200离心5min，弃上清；
(6)每管加入300μl的Wash Buffer重悬细胞，并转移至流式管中，用流式细胞仪检测荧光值。
3.实验结果
如图10-1所示。
结果显示，相较于Mock T细胞，其余细胞各种细胞因子(IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ)的分泌量都有很大的提高。Mock T各种细胞因子的分泌量最弱，meso G1 CAR各种细胞因子的分泌量较弱，meso G2 CAR各种细胞因子的分泌量较强，meso G1 CAR-137DCR1各种细胞因子的分泌量最强。
实施例7-(2)：流式检测Muc1抗原刺激下Muc1 CAR-T细胞因子分泌量
1.实验样品
实施例2-(2)制得的重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR1和Mock T。
2.实验方法
1.用5μg/ml的Muc1抗原包被96孔板，4℃包被过夜，PBS清洗3遍，分别加入1×10
5的各样品细胞，培养24h后收集细胞上清。用BD
TMCBA Human Th1/Th2 Cytokine Kit II检测Muc1 CAR-T细胞受Muc1抗原刺激后细胞因子的分泌情况。
具体步骤如下：
(1)混合人的IL-2、IL-4、IL-6、IL-10、TNF、IFN-γ捕获磁珠，涡旋振荡混匀捕获磁珠，每管加入50μl混匀后的捕获磁珠；
(2)加入50μl人的Th1/Th2细胞因子标准品(倍比稀释5000pg/ml、2500pg/ml、1250pg/ml、625pg/ml、312.5pg/ml、156pg/ml、80pg/ml、40pg/ml、20pg/ml、0pg/ml)和50μl的待测样品(经稀释液2倍稀释)。
(3)每管加入50μl的人的Th1/Th2-II-PE的检测抗体；
(4)室温避光孵育3h；
(5)每管加入1ml的Wash Buffer，200离心5min，弃上清；
(6)每管加入300μl的Wash Buffer重悬细胞，并转移至流式管中，用流式细胞仪检测荧光值。
3.实验结果
如图10-2所示。
结果显示，相较于Mock T细胞，其余细胞各种细胞因子(IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ)的分泌量都有很大的提高。Mock T各种细胞因子的分泌量最弱，Muc1 G1 CAR各种细胞因子的分泌量较弱，Muc1 G2 CAR各种细胞因子的分泌量较强，Muc1 G1 CAR-28DCR1各种细胞因子的分泌量最强。
实施例7-(3)：流式检测EGFR抗原刺激下EGFR CAR-T细胞因子分泌量
1.实验样品
实施例2-(3)制得的重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR1和Mock T。
2.实验方法
1.用5μg/ml的EGFR抗原包被96孔板，4℃包被过夜，PBS清洗3遍，分别加入1×10
5的各样品细胞，培养24h后收集细胞上清。用BD
TMCBA Human Th1/Th2 Cytokine Kit II检测EGFR CAR-T细胞受EGFR抗原刺激后细胞因子的分泌情况。
具体步骤如下：
(1)混合人的IL-2、IL-4、IL-6、IL-10、TNF、IFN-γ捕获磁珠，涡旋振荡混匀捕获磁珠，每管加入50μl混匀后的捕获磁珠；
(2)加入50μl人的Th1/Th2细胞因子标准品(倍比稀释5000pg/ml、2500pg/ml、1250pg/ml、625pg/ml、312.5pg/ml、156pg/ml、80pg/ml、40pg/ml、20pg/ml、0pg/ml)和50μl的待测样品(经稀释液2倍稀释)。
(3)每管加入50μl的人的Th1/Th2-II-PE的检测抗体；
(4)室温避光孵育3h；
(5)每管加入1ml的Wash Buffer，200离心5min，弃上清；
(6)每管加入300μl的Wash Buffer重悬细胞，并转移至流式管中，用流式细胞仪检测荧光值。
3.实验结果
如图10-3所示。
结果显示，相较于Mock T细胞，其余细胞各种细胞因子(IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ)的分泌量都有很大的提高。Mock T各种细胞因子的分泌量最弱，EGFR G1 CAR各种细胞因子的分泌量较弱，EGFR G2 CAR各种细胞因子的分泌量较强，EGFR G1 CAR-40DCR1各种细胞因子的分泌量最强。
实施例8-(1):双向共刺激分子激活受体137DCR联合meso G1 CAR-T细胞的体
内功能实验
1.实验样品和实验动物
4－6周龄NSG完全免疫缺陷小鼠20只，平均重量22-27g，由北京维通达生物技术有限公司提供，SPF级动物实验室饲养。
实施例2-(1)制得的重组细胞meso G1 CAR、meso G2 CAR、meso G1 CAR-137DCR3和Mock T。
卵巢癌细胞SK-OV-3-luc(购自上海慧颖生物科技有限公司)。
2.实验方法
(1)体外培育人卵巢癌细胞SK-OV-3-luc，取对数生长期贴壁生长细胞，0.25％胰酶消化，离心、收集细胞后用PBS液重悬，1000rmp室温离心2分钟，弃上清，再用PBS液重悬后离心收集细胞，调整细胞悬液浓度至5×10
7个/ml。
(2)于小鼠右肋背部皮下接种SK-OV-3-luc细胞，0.1ml/只。接种10天后，可通过活体成像仪观察肿瘤大小。
(3)将NSG免疫缺陷小鼠随机分为4组，每组5只。给药途径为直接尾静脉注射，每只0.1ml/只，即5×10
6个阳性细胞，溶剂为PBS。只给药一次。
(4)每日观察小鼠的生活状态并每隔10天通过活体成像仪观察小鼠肿瘤变化。
3.实验结果
如图11-1所示。
结果显示，Mock T是对照组，肿瘤细胞荧光强度强，meso G1 CAR是靶向间皮素的一代CAR，肿瘤细胞荧光强度减弱，表明有一定的治疗效果，meso G2 CAR肿瘤细胞荧光强度更弱，表明有更好的治疗效果，meso G1 CAR-137DCR3肿瘤细胞荧光强度最弱，表明有最好的治疗效果。
实施例8-(2):双向共刺激分子激活受体28DCR联合Muc1 G1 CAR-T细胞的体
内功能实验
1.实验样品和实验动物
4－6周龄NSG完全免疫缺陷小鼠20只，平均重量22-27g，由北京维通达生物技术有限公司提供，SPF级动物实验室饲养。
实施例2-(2)制得的重组细胞Muc1 G1 CAR、Muc1 G2 CAR、Muc1 G1 CAR-28DCR3和Mock T。
卵巢癌细胞SK-OV-3-luc(购自上海慧颖生物科技有限公司)。
2.实验方法
(1)体外培育人卵巢癌细胞SK-OV-3-luc，取对数生长期贴壁生长细胞，0.25％胰酶消化，离心、收集细胞后用PBS液重悬，1000rmp室温离心2分钟，弃上清，再用PBS液重悬后离心收集细胞，调整细胞悬液浓度至5×10
7个/ml。
(2)于小鼠右肋背部皮下接种SK-OV-3-luc细胞，0.1ml/只。接种10天后，可通过活体成像仪观察肿瘤大小。
(3)将NSG免疫缺陷小鼠随机分为4组，每组5只。给药途径为直接尾静脉注射，每只0.1ml/只，即5×10
6个阳性细胞，溶剂为PBS。只给药一次。
(4)每日观察小鼠的生活状态并每隔10天通过活体成像仪观察小鼠肿瘤变化。
3.实验结果
如图11-2所示。
结果显示，Mock T是对照组，肿瘤细胞荧光强度强，Muc1 G1 CAR是靶向Muc1的一代CAR,肿瘤细胞荧光强度减弱，表明有一定的治疗效果，Muc1 G2 CAR肿瘤细胞荧光强度更弱，表明有进一步的好的治疗效果，Muc1 G1 CAR-28DCR3肿瘤细胞荧光强度最弱，表明有最好的治疗效果。
实施例8-(3):双向共刺激分子激活受体40DCR联合EGFR G1 CAR-T细胞的体
内功能实验
1.实验样品和实验动物
4－6周龄NSG完全免疫缺陷小鼠20只，平均重量22-27g，由北京维通达生物技术有限公司提供，SPF级动物实验室饲养。
实施例2-(3)制得的重组细胞EGFR G1 CAR、EGFR G2 CAR、EGFR G1 CAR-40DCR3和Mock T。
卵巢癌细胞SK-OV-3-luc(购自上海慧颖生物科技有限公司)。
2.实验方法
(1)体外培育人卵巢癌细胞SK-OV-3-luc，取对数生长期贴壁生长细胞，0.25％胰酶消化，离心、收集细胞后用PBS液重悬，1000rmp室温离心2分钟，弃上清，再用PBS液重悬后离心收集细胞，调整细胞悬液浓度至5×10
7个/ml。
(2)于小鼠右肋背部皮下接种SK-OV-3-luc细胞，0.1ml/只。接种10天后，可通过活体成像仪观察肿瘤大小。
(3)将NSG免疫缺陷小鼠随机分为4组，每组5只。给药途径为直接尾静脉注射，每只0.1ml/只，即5×10
6个阳性细胞，溶剂为PBS。只给药一次。
(4)每日观察小鼠的生活状态并每隔10天通过活体成像仪观察小鼠肿瘤变化。
3.实验结果
如图11-3所示。
结果显示，Mock T是对照组，肿瘤细胞荧光强度强，EGFR G1 CAR是靶向EGFR的一代CAR,肿瘤细胞荧光强度减弱，表明有一定的治疗效果，EGFR G2 CAR肿瘤细胞荧光强度更弱，表明有进一步的好的治疗效果，EGFR G1 CAR-40DCR3肿瘤细胞荧光强度最弱，表明有最好的治疗效果。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (20)
- 一种分离的多肽，其从N端到C端依次包括下述元件：可选的信号肽、激活共刺激信号分子的多肽(例如共刺激信号分子的激活型单链抗体或共刺激信号分子的配体)、胞外铰链区、跨膜区和胞内共刺激信号分子。
- 根据权利要求1所述的多肽，其特征在于如下的(1)－(5)项中的任意1项、2项、3项、4项或者5项：(1)所述信号肽为膜蛋白信号肽；优选地，所述信号肽为选自CD8信号肽、CD28信号肽和CD4信号肽中的一种或多种；优选地，所述信号肽是CD8信号肽；优选地，CD8信号肽的氨基酸序列如SEQ ID NO:1所示；(2)所述共刺激信号分子的激活型单链抗体选自CD137激活型单链抗体、CD28激活型单链抗体和CD40激活型单链抗体中的任意一种或者多种；所述共刺激信号分子的配体选自CD137的配体、CD28的配体和CD40的配体中的任意一种或多种；优选地，所述CD137激活型单链抗体的氨基酸序列如SEQ ID NO:2所示；优选地，所述CD28激活型单链抗体的氨基酸序列如SEQ ID NO:31所示；优选地，所述CD40激活型单链抗体的氨基酸序列如SEQ ID NO:55所示；优选地，所述CD137的配体是4-1BBL；优选地，所述CD28的配体是CD80/CD86；优选地，所述CD40的配体是CD40L；(3)所述胞外铰链区为选自IgG4Fc CH2CH3铰链区、CD28铰链区和CD8铰链区的一种或多种；优选地，为CD8铰链区；优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:3所示；优选地，为IgG4Fc CH2CH3铰链区；优选地，所述IgG4Fc CH2CH3铰链区的氨基酸序列如SEQ ID NO:56所示；(4)所述跨膜区为选自CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种；优选地，所述跨膜区为CD28跨膜区；优选地，所述CD28跨膜区的氨基酸序列如SEQ ID NO:4所示；(5)所述胞内共刺激信号分子选自CD28胞内结构域、CD134/OX40胞内结构域、 CD137/4-1BB胞内结构域、LCK胞内结构域、ICOS胞内结构域和DAP10胞内结构域中的一种或多种；优选地，所述胞内共刺激信号分子为CD28胞内结构域和/或CD137胞内结构域；优选地，所述CD28胞内结构域的氨基酸序列如SEQ ID NO:5所示；优选地，所述CD137胞内结构域的氨基酸序列如SEQ ID NO:6所示。
- 根据权利要求1或2所述的多肽，其从N端到C端依次包括下述元件：可选的CD8信号肽、CD137激活型单链抗体、CD8胞外铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域；可选的CD8信号肽、CD28激活型单链抗体、CD8胞外铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域；或者可选的CD8信号肽、CD40激活型单链抗体、IgG4Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和/或CD137胞内结构域。
- 根据权利要求1至3中任一权利要求所述的多肽，其氨基酸序列如SEQ ID NO:7至SEQ ID NO:14中任一序列所示；SEQ ID NO:32至SEQ ID NO:39中任一序列所示；或者SEQ ID NO:57至SEQ ID NO:64中任一序列所示。
- 一种分离的多核苷酸，其编码权利要求1至4中任一权利要求所述的分离的多肽；优选地，所述分离的多核苷酸的序列如SEQ ID NO:15或SEQ ID NO:22中任一序列所示；SEQ ID NO:40至SEQ ID NO:47中任一序列所示；或者SEQ ID NO:65至SEQ ID NO:72中任一序列所示。
- 一种核酸构建体，包含权利要求5所述的多核苷酸。
- 一种重组载体，其含有权利要求5所述的多核苷酸或者权利要求6所述的核酸构建体；优选地，所述重组载体为重组克隆载体、重组真核表达质粒或者重组病毒载体；优选地，所述重组表达载体为重组的转座子载体；优选地，所述转座子载体含有选自piggybac、sleeping beauty、frog prince、Tn5或Ty的转座元件；优选地，所述 重组表达载体为权利要求5所述的多核苷酸与PS328b载体经重组得到的重组载体。
- 一种重组载体组合，其包含第一重组载体和第二重组载体，其中：所述第一重组载体为权利要求7所述的重组载体，所述第二重组载体含有第一代嵌合抗原受体的编码序列；优选地，所述第一代嵌合抗原受体为靶向间皮素、Muc1或EGFR的第一代嵌合抗原受体；优选地，所述第一代嵌合抗原受体的氨基酸序列如SEQ ID NO:23、SEQ ID NO:48或SEQ ID NO:73所示；优选地，所述第一代嵌合抗原受体的核酸序列如SEQ ID NO:24、SEQ ID NO:49或SEQ ID NO:74所示；优选地，所述第二重组载体为重组的PNB328载体。
- 一种重组宿主细胞，其中，所述细胞含有权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体或者权利要求8所述的重组载体组合；优选地，所述重组宿主细胞为重组哺乳动物细胞；优选地，所述重组宿主细胞为重组T细胞；优选地，所述重组T细胞为重组的外周血单核细胞。
- 一种T细胞，其表达有权利要求1至4中任一权利要求所述的多肽，以及第一代嵌合抗原受体；优选地，所述重组T细胞为重组的外周血单核细胞；优选地，所述第一代嵌合抗原受体为靶向间皮素、Muc1或EGFR的第一代嵌合抗原受体；优选地，所述第一代嵌合抗原受体的氨基酸序列如SEQ ID NO:23、SEQ ID NO:48或SEQ ID NO:73所示。
- 一种药用组合物，其包含权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞；可选地，还包含药学上可接受的辅料。
- 权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞在制备治疗和/或 预防癌症的药物中的用途；优选地，所述癌症为其癌细胞表面异常表达间皮素、Muc1或EGFR的癌症；优选地，所述癌症选自：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞在制备抑制癌细胞的药物中的用途；优选地，所述癌细胞为细胞表面异常表达间皮素、Muc1或EGFR的癌细胞；优选地，所述癌细胞选自如下癌症的癌细胞：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞在制备促进细胞因子分泌的药物中的用途，其中，所述细胞因子选自IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ中的一种或多种。
- 根据权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞，其用于治疗和/或预防癌症；优选地，所述癌症为其癌细胞表面异常表达间皮素、Muc1或EGFR的癌症；优选地，所述癌症选自：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 根据权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞，其用于抑制癌细胞；优选地，所述癌细胞为细胞表面异常表达间皮素、Muc1或EGFR的癌细胞；优选地，所述癌细胞选自如下癌症的癌细胞：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 根据权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞，其用于促进细胞因子分泌，其中，所述细胞因子选自IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ中的一种或多种。
- 一种治疗和/或预防癌症的方法，包括给予有需求的受试者以有效量的权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞的步骤；优选地，所述癌症为其癌细胞表面异常表达间皮素、Muc1或EGFR的癌症；优选地，所述癌症选自：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 一种在体内或在体外抑制癌细胞的方法，包括施加癌细胞以有效量的权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞的步骤；优选地，所述癌细胞为细胞表面异常表达间皮素、Muc1或EGFR的癌细胞；优选地，所述癌细胞选自如下癌症的癌细胞：腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌或前列腺癌。
- 一种在体内或在体外促进T细胞的细胞因子分泌的方法，包括施加T细胞以有效量的权利要求1至4中任一权利要求所述的多肽、权利要求5所述的多核苷酸、权利要求6所述的核酸构建体、权利要求7所述的重组载体、权利要求8所述的重组载体组合、权利要求9所述的重组宿主细胞或者权利要求10所述的T细胞的步骤；其中，所述细胞因子选自IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ中的一种或多种。
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| CN116640226B (zh) * | 2022-04-02 | 2024-04-26 | 广东东阳光药业股份有限公司 | 一种抑制宿主抗外源免疫细胞hvg反应的嵌合受体 |
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| EP3733695A4 (en) | 2021-12-22 |
| JP7386177B2 (ja) | 2023-11-24 |
| JP2021509290A (ja) | 2021-03-25 |
| CN109970864A (zh) | 2019-07-05 |
| EP3733695A1 (en) | 2020-11-04 |
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