CN109971713B

CN109971713B - Muc 1-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof

Info

Publication number: CN109971713B
Application number: CN201711457432.5A
Authority: CN
Inventors: 钱其军; 金华君; 何周; 李林芳; 刘祥箴; 王超; 崔连振
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2023-06-20
Anticipated expiration: 2037-12-28
Also published as: WO2019128994A1; CN109971713A

Abstract

The invention relates to CAR-T cells specifically targeting Muc1 antigen and stably expressing PD-1 antibodies at high levels and uses thereof. Specifically, the T cells of the invention contain coding sequences for expressing chimeric antigen receptors that recognize Muc1 antigen and coding sequences for PD-1 antibodies; and/or express chimeric antigen receptors that recognize Muc1 antigen and PD-1 antibodies. The chimeric antigen receptor provided by the invention sequentially comprises a membrane protein signal peptide, an anti-Muc 1 near-membrane single-chain antibody, a hinge region with more than 50 amino acid residues in length, a transmembrane region, a co-stimulatory signal molecule intracellular domain and an immune receptor tyrosine activation motif from the N end to the C end. The T cell provided by the invention can overcome the inhibition of immune microenvironment, promote the apoptosis of tumor cells and exert anti-tumor immune response, and can be used for the treatment of various Muc1 positive malignant tumors.

Description

Muc 1-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof

Technical Field

The present invention relates to Muc 1-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof.

Background

The chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) treatment technology is certainly a very rising giant star in the tumor immune cell treatment field. The CAR-T technology is characterized in that after the gene sequence of an antibody variable region for recognizing a certain antigen molecule is spliced with the sequence of an intracellular region of a T lymphocyte immune receptor by a genetic engineering technology, the antigen variable region is transduced into lymphocytes by a retrovirus or lentiviral vector, a transposon or a transposase system or direct mRNA, and fusion proteins are expressed on the surfaces of the cells, so that the T lymphocytes can recognize specific antigens in a non-MHC (major histocompatibility) restriction mode, and the tumor recognition and killing capacity of the T lymphocytes is enhanced.

The structure of the CAR is first proposed by the Saint Eshhar research group in 1989, and after development for nearly 30 years, it has been proved that T cells modified by the CAR structure have better curative effects in tumor immunotherapy. The first generation of CAR receptors contained a fragment (single-chain variable fragment, scFv) that specifically recognized tumor antigens extracellular, with intracellular activation signals transmitted by the cd3ζ signal chain. However, the first generation of CAR receptors lack co-stimulatory signals from T cells, resulting in T cells that can only exert transient effects, short in vivo, and low cytokine secretion. The second generation CAR receptor increases the intracellular domains of costimulatory signaling molecules, including domains such as CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory (ICOS) and DNAX activating protein 10 (DAP 10), enhancing proliferation capacity of T cells and secretion function of cytokines, and increases IL-2, IFN- γ and GM-CSF, thereby breaking through immunosuppression of tumor microenvironment, prolonging AICD (activation-induced cell death, AICD). The third generation CAR receptor fuses a secondary co-stimulatory molecule such as 4-1BB between the co-stimulatory structure CD28 and the ITAM signal chain, thereby generating a triple-signaling CAR receptor, and the T cells modified by the third generation CAR receptor have better effector function and in vivo survival time. The conventional classical CAR-T structure is a second-generation CAR receptor, and the structure can be specifically divided into the following 4 parts: an antibody single chain variable region (scFv), a hinge region, a transmembrane region, and an intracellular stimulatory signaling region that recognizes a tumor antigen. Wherein the CAR structure hinge region is responsible for forming the correct conformation and dimer. The length of the hinge region and the amino acid sequence characteristics determine the spatial conformation of the CAR and also its ability to bind to tumor cell surface antigens.

Solid tumors have high heterogeneity, and have high differences among different patients, different focuses of the same patient and different tumor cells of the same focus. This high degree of heterogeneity renders tumor targeted therapies lacking ideal universal, broad-spectrum targets, limiting the efficacy of CAR-T cell therapies for solid tumors. Thus, finding an effective CAR-T cell therapeutic target has become a serious issue in CAR-T cell therapy. Muc1 (mucin) is a type I transmembrane glycoprotein with high molecular weight (> 200 kD) and is normally mainly expressed in various tissues and organs by the epithelial cells near lumen or glandular lumen surface, expressed in top and distributed in polarity, and the Ser/Thr on the polypeptide skeleton is connected with O-glycosidic bond. When the tumor occurs, the Muc1 protein can be abnormally expressed on the surface of tumor cells, and the expression quantity can reach more than 100 times that of the normal tumor cells. Moreover, the polar distribution on the cell surface is lost, and the polar distribution can be uniformly distributed on the whole cell surface. In addition, the structure of Muc1 protein is also changed due to the glycosylation insufficiency, and new sugar chains and peptide epitopes are present. Although these new sugar chains and peptide epitopes may be ideal targets for CAR-T cell therapy, CAR-T cells that specifically target a certain glycopeptide may not find use in many types of tumors because they may exhibit different sugar chains and peptide epitopes on different tumor cell surfaces.

PD-1 (Programmed Death 1, reprogrammed cell Death receptor 1) is a member of the CD28 family of regulatory T cells belonging to the immunoglobulin superfamily of receptors. PD-1 and its ligand PD-L1/PD-L2 play an important role in co-suppression and depletion of T cells, their interactions inhibit co-stimulatory molecule-mediated proliferation of T cells and cytokine secretion, down-regulate expression of anti-apoptotic molecule BCL-xl, impair tumor-specific T cell function, and result in some tumor patients not being able to completely eliminate tumors. The PD-1 antibody competes for the binding of PD-1 to its ligand PD-L1/PD-L2 by binding to the PD-1 molecule on the surface of tumor-specific T cells, thereby alleviating the immune microenvironment inhibition caused by the binding of PD-1 to PD-L1/PD-L2. Currently commercialized PD-1 antibodies are Nivolumab and Pidilizumab. The 2 monoclonal antibodies have proved to have good clinical effects on solid tumors such as melanoma, colon cancer, prostate cancer, non-small cell lung cancer, renal cell carcinoma and the like, but the PD-1 antibodies still have some unavoidable problems in clinical application. On the one hand, since PD-1 monoclonal antibody is used for intravenous injection and systemic administration, most patients receiving PD-1 antibody blocking treatment have different degrees of drug toxic and side effects. On the other hand, the production of PD-1 monoclonal antibody involves complex production, preparation and purification processes, and has high cost, so that the treatment cost is high.

In conclusion, the CAR-T cells have the capability of killing tumor cells and can effectively enter the tumor tissues, but the activity of the CAR-T cells is easy to be inhibited in the tumor microenvironment; the PD-1 antibody can reactivate the anti-tumor activity of T cells, but the penetration of the macromolecular antibody to solid tumors is insufficient, and the systemic administration has larger toxic and side effects and high drug cost. Therefore, if the PD-1 antibody can be efficiently expressed by the CAR-T cell under the premise of keeping the killing toxicity of the CAR-T cell, the PD-1 antibody can be expressed at a local high level on the tumor by the tumor trend characteristic of the CAR-T cell, so that the defects of the CAR-T cell treatment and the PD-1 antibody treatment can be overcome simultaneously, the synergistic effect of the CAR-T cell treatment and the PD-1 antibody treatment can be exerted, the curative effect can be improved, and the treatment cost can be reduced.

Disclosure of Invention

The present invention provides a T cell that: (1) Comprising coding sequences for a chimeric antigen receptor that recognizes the Muc1 antigen and coding sequences for PD-1 antibodies; and/or (2) express chimeric antigen receptor and PD-1 antibody that recognize Muc1 antigen.

In one or more embodiments, the T cell genome incorporates an expression cassette for a chimeric antigen receptor that recognizes the Muc1 antigen and an expression cassette for a PD-1 antibody.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a membrane protein signal peptide, a single chain antibody against the near membrane terminus of Muc1, a hinge region of more than 50 amino acid residues in length, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

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

In one or more embodiments, the amino acid sequence of the single chain antibody is shown in SEQ ID NO. 7.

In one or more embodiments, the hinge region of greater than 50 amino acid residues in length is selected from the group consisting of a CD8 a hinge region, an IgD hinge region, an IgG1Fc CH2CH3 hinge region, and an IgG4Fc CH2CH3 hinge region; preferably, the hinge region is a CD8 a hinge region or an IgG4Fc CH2CH3 hinge region; more preferably, the amino acid sequence of the CD 8. Alpha. Hinge region is shown in SEQ ID NO. 8; the amino acid sequence of the IgG4Fc CH2CH3 hinge region is shown in SEQ ID NO. 3.

In one or more embodiments, the transmembrane region is one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably CD28, and preferably has the amino acid sequence shown in SEQ ID NO. 9.

In one or more embodiments, the intracellular co-stimulatory signaling domain includes an intracellular domain of a co-stimulatory signaling molecule, including an intracellular domain of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS), and DNAX activator protein 10; preferably, the intracellular co-stimulatory signaling domain is the intracellular domain of CD 28; preferably, the amino acid sequence of the CD28 is shown in SEQ ID NO. 10.

In one or more embodiments, the intracellular signaling domain is a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3ζ intracellular signaling domain, preferably the amino acid sequence of said CD3ζ intracellular signaling domain is depicted in SEQ ID NO. 12.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-Muc 1 single chain antibody, a CD8a hinge region or IgG4Fc CH2CH3 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and a tyrosine activation motif of cd3ζ.

In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-Muc 1 single chain antibody, an IgG4Fc CH2CH3 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and a tyrosine-activating motif of cd3ζ.

In one or more embodiments, the chimeric antigen receptor has an amino acid sequence as shown in amino acid residues 23-678 of SEQ ID NO. 13, or as shown in SEQ ID NO. 13; preferably, the coding sequence of the chimeric antigen receptor is shown as the 70 th to 2034 th bases of SEQ ID NO. 14 or as the SEQ ID NO. 14.

In one or more embodiments, the PD-1 antibody has an amino acid sequence as set forth in amino acid residues 21-495 of SEQ ID NO. 1, or as set forth in SEQ ID NO. 1; preferably, the coding sequence of the PD-1 antibody is shown as 61-1488 bases of SEQ ID NO. 2 or as shown in SEQ ID NO. 2.

The present invention also provides a composition comprising: a vector comprising an expression cassette for a chimeric antigen receptor of the invention for integration of the expression cassette into the genome of a host cell; and a vector comprising an expression cassette for a PD-1 antibody, said vector for integration of said expression cassette into the genome of a host cell.

In one or more embodiments, the PD-1 antibody has an amino acid sequence as set forth in amino acid residues 21-495 of SEQ ID NO. 1, or as set forth in SEQ ID NO. 1; preferably, the coding sequence of the PD-1 antibody is shown as the 63 st to 1488 th bases of SEQ ID NO. 2 or as the SEQ ID NO. 2.

The invention also provides a kit comprising:

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

(2) A vector comprising an expression cassette for a PD-1 antibody, said vector being used to integrate said expression cassette into the genome of a host cell.

In one or more embodiments, the PD-1 antibody has an amino acid sequence as set forth in amino acid residues 21-495 of SEQ ID NO. 1.

The invention also provides a pharmaceutical composition containing the T cells or the T cells and the PD-1 antibodies expressed by the T cells.

The invention also provides the use of the T cells described herein or of the T cells and their expressed PD-1 antibodies in the manufacture of a medicament for the treatment or prophylaxis of malignant tumors. Preferably, the cancer is a cancer in which Muc1 is abnormally expressed on the surface of cancer cells, preferably a cancer in which the expression amount of Muc1 on the surface of cancer cells is 100 times or more than that when the expression amount is normal, and in which Muc1 is uniformly distributed on the whole cell surface; preferably, the cancer is selected from: adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer, or prostate cancer.

Drawings

Fig. 1: pNB328-Muc1CAR, pS328-Muc1CAR, pNB328-m279V, pS328-m279V-wt, pS328-m279V, pNB-Muc 1CAR-2A-m279V,

Expression cassette pattern of pNB328-m279V-IRES-Muc1 CAR.

Fig. 2A-2B: and (3) determining the positive rate and the expression quantity of the expressed Muc1CAR gene and the PD-1 antibody of the PBMCs after the modification and activation of different combination forms of the Muc1CAR gene and the PD-1 antibody gene.

Fig. 3A-3B: and (3) determining the positive rate and the PD-1 antibody expression quantity of the gene of the modified PBMCs by different mass ratios of pNB328-Muc1CAR to pS328-m279V plasmids.

Fig. 4: the positive rate of the Muc1CAR gene expressed by the T cells after the modification and activation of the Muc1CAR gene and the PD-1 antibody gene of the PBMCs from different patient sources (patient 1, patient 2 and patient 3 in sequence from top to bottom) is measured.

Fig. 5: ELISA assays of PD-1 antibody Gene expression of different patient-derived PBMCs modified with the PD-1 antibody Gene.

Fig. 6A-6D: muc1CAR-anti PD1 pluripotent T cells can enhance the killing activity of the T cells in vitro. A: the flow assay indirectly reflects the T cell killing activity of the marker CD107 alpha, the activation marker CD25 and the depletion marker LAG3, B the flow assay reflects the T cell memory phenotype of the marker proteins CD45RO, CD62L and CCR7.C: the ratio of T cells CD3/CD4/CD8 was flow tested. D: and detecting the change of IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma cytokines of the Muc1CAR-anti PD1 pluripotent T cells, the Muc1CAR-T cells and the Mock T cells after the Muc1 antigen is stimulated by the multi-factor detection kit.

Fig. 7: detection of killing by Muc1CAR-anti PD1 pluripotent T cells.

Fig. 8: in vivo functional studies of Muc1CAR T cells expressing PD-1 antibodies.

Detailed Description

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

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

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

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

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

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

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

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

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

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

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

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

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

Studies show that the IgG4Fc fragment of the PD-1 antibody is easy to be recognized by mononuclear/macrophages and phagocytosed, and the PD-1 antibody can well perform and not cause ADCC reaction when the PD-1 antibody IgG4Fc fragment is subjected to base mutation modification so as to meet the requirement of the PD-1 antibody expressed by T cells.

Accordingly, the present invention provides a PD-1 antibody comprising an anti-PD-1 single chain antibody and an IgG4Fc. In certain embodiments, the amino acid sequence of the IgG4Fc is shown as amino acid residues 267-495 of SEQ ID NO. 1; preferably, the coding sequence is shown as the base sequence of 799-1485 of SEQ ID NO. 2.

In certain embodiments, the anti-PD-1 single chain antibody (scFv) has an antibody light chain variable region (VL region) amino acid sequence as set forth in amino acid residues 21-131 of SEQ ID NO. 1; preferably, the coding sequence is shown as 61-393 base sequences of SEQ ID NO. 2. In certain embodiments, the heavy chain variable region (VH region) amino acid sequence of the anti-PD-1 single-chain antibody is shown as 147-266 amino acid sequences of SEQ ID NO. 1; preferably, the coding sequence is shown as the base sequence of 439-798 of SEQ ID NO. 2. In certain embodiments, the anti-PD-1 single-chain antibody has an amino acid sequence as set forth in amino acid residues 21-266 of SEQ ID NO. 1; preferably, the coding sequence is shown as 61-798 base sequences of SEQ ID NO. 2.

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

In certain embodiments, the PD-1 antibody has an amino acid sequence as set forth in SEQ ID NO. 1 at amino acid positions 21-495 or as set forth in SEQ ID NO. 1.

The invention also includes the coding sequence of the PD-1 antibody or its complement, which comprises at least the coding sequence of IgG4Fc described herein or its complement. In certain embodiments, the coding sequence of the PD-1 antibody comprises the sequence set forth in base sequence positions 61-1495 of SEQ ID NO. 2, preferably the sequence set forth in SEQ ID NO. 2.

The invention also includes a nucleic acid construct comprising the coding sequence of the PD-1 antibodies of the invention or the complement thereof. Preferably, the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or the complement thereof into a host cell.

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

The invention also provides the use of the PD-1 antibodies, their coding sequences or complementary sequences, nucleic acid constructs, and host cells in the preparation of a method for treating or preventing a malignancy, particularly a PD-1-associated neoplasm, including, but not limited to, the various malignancies described herein.

There are several ways to express 2 different proteins in the same cell, including 2A or IRES ligation of 2 gene fragments to form a single plasmid followed by in vitro modification of the cell, which ensures that one gene must be expressed in the same cell after the other gene is expressed in a cell, but that the gene fragment carried by the plasmid if too long results in weaker expression of the transcribed and translated protein. Alternatively, 2 gene fragments may be constructed on 2 vectors, respectively, while modifying the cells in vitro. This method can enhance the expression of the transcribed and translated protein, but cannot ensure that 2 proteins are expressed in the same cell.

In order to realize that both the Muc1CAR gene and the PD-1 antibody can be expressed efficiently and stably in cells, the invention performs various combination forms of tests on the Muc1CAR gene and the PD-1 antibody, wherein the tests comprise that the Muc1CAR gene, the PD-1 antibody and the PB gene are connected by 2A to form a single plasmid, the Muc1CAR gene is connected by IRES to form a single plasmid with the PD-1 antibody and the PB gene, the double plasmid combination of the Muc1CAR gene plasmid carrying the PB gene and the PD-1 antibody plasmid and the double plasmid combination of the PD-1 antibody plasmid carrying the PB gene and the Muc1CAR gene plasmid. Tests show that the combination of the Muc1CAR gene plasmid carrying the PB gene and the double plasmids of the PD-1 antibody plasmid can obtain stable expression of the Muc1CAR gene and the PD-1 antibody.

Therefore, the invention also provides a T cell modified by the Muc1CAR gene and capable of expressing the PD-1 antibody, the T cell can stably express the Muc1CAR gene and the PD-1 antibody at a high level, the externally expressed Muc1CAR gene can accurately target the Muc1 antigen, the proliferation capacity and the secretion of cytokines of the T cell are enhanced, the killing of the CAR-T cell on the tumor cell is enhanced, and the anti-tumor effect is exerted by enhancing the immune response. Meanwhile, the exogenously expressed PD-1 antibody can overcome the inhibition of immune microenvironment, promote the apoptosis of tumor cells and play an anti-tumor immune response. In addition, the exogenous Muc1CAR gene and PD-1 antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stabilizing sustained expression in T cells. The T cells capable of stably expressing the Muc1CAR gene and the PD-1 antibody gene at high level can be used for treating various malignant tumors with high Muc1 expression.

The CARs of the invention generally contain an optional signal peptide sequence, an scFv that recognizes the Muc1 antigen, a hinge region, a transmembrane region, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

The signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of a newly synthesized protein to the secretory pathway, often referred to as the N-terminal amino acid sequence (sometimes not necessarily at the N-terminus) of the newly synthesized polypeptide chain that directs the transmembrane transfer (localization) of the protein, which is responsible for directing the protein into subcellular organelles of the cell containing different membrane structures. The signal peptide may be a secretory signal peptide or a membrane-bound signal peptide. In certain embodiments of the invention, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, or a CD4 signal peptide; more preferably a CD8 signal peptide. The amino acid sequence of the CD8 signal peptide can be shown as SEQ ID NO. 5; in certain embodiments, the coding sequence is shown as bases 1-66 of SEQ ID NO. 14.

The scFv that recognizes the Muc1 antigen described herein may be a single chain antibody directed against the Muc1 antigen as known in the art. Preferably, the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody directed against the membrane proximal amino acid sequence of Muc 1. In certain embodiments, the Muc1 membrane proximal amino acid sequence is shown in SEQ ID NO. 6. An exemplary anti-Muc 1 single chain antibody has an amino acid sequence shown in SEQ ID NO. 7, and an exemplary coding sequence shown in SEQ ID NO. 14 at bases 67-807.

The hinge region, as used herein, refers to the region between the functional regions of the heavy chains CH1 and CH2 of an immunoglobulin which is rich in proline, does not form an alpha helix, and is subject to stretching and some degree of warping, which facilitates complementary binding between the antigen binding site of the antibody and the epitope. Hinge regions suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8a, the IgG1FcCH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4Fc CH2CH3 hinge region, and the extracellular hinge region of CD 4. The hinge region is preferably a hinge region that is more than 50 amino acid residues in length, more preferably more than 80 amino acids in length. In certain embodiments, a CD8a hinge region or an IgG4FcCH2CH3 hinge region is used herein. An exemplary CD8a hinge region has the amino acid sequence set forth in SEQ ID NO: shown at 8. The amino acid sequence of an exemplary IgG4FcCH2CH3 hinge region is shown in SEQ ID NO. 3, and the coding sequence of an exemplary IgG4FcCH2CH3 hinge region is shown in SEQ ID NO. 4 or in SEQ ID NO. 14 at positions 808-1491.

The transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a cd3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD28 transmembrane region, preferably having the amino acid sequence shown in SEQ ID NO. 9; in certain embodiments, the coding sequence is shown as SEQ ID NO. 14 at bases 1492-1575.

Intracellular costimulatory signaling domains the intracellular domain comprising the costimulatory signaling molecule may be selected from the group consisting of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory factor (ICOS) and DNAX activator protein 10 (DAP 10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably having the amino acid sequence shown in SEQ ID NO. 10, and exemplary coding sequences are shown in SEQ ID NO. 14 at bases 1576-1698. In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD137/4-1 BB; preferably, the amino acid sequence of the CD137/4-1BB is shown as SEQ ID NO. 11.

The intracellular signaling domain is preferably an immunoreceptor tyrosine-activating motif, which may be a cd3ζ intracellular signaling domain or an fcsriy intracellular signaling domain; preferably a CD3ζ intracellular signaling domain, preferably the amino acid sequence of said CD3ζ intracellular signaling domain is depicted in SEQ ID NO. 12; in certain embodiments, the coding sequence is as shown in SEQ ID NO. 10 at bases 1699-2034.

In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: optionally a CD signal peptide, scFv, igG4Fc CH2CH3 hinge region, CD28 transmembrane region, intracellular domain of CD28, and cd3ζ intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is shown as amino acid residues 23-678 of SEQ ID NO. 13. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 13. In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: optionally a CD signal peptide, scFv, a CD8 a hinge region, a CD28 transmembrane region, an intracellular domain of CD28 and a cd3ζ intracellular signal domain.

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

The above-described portions forming the chimeric antigen receptor herein, such as the signal peptide, the light chain variable region and heavy chain variable region of the anti-Muc 1 single chain antibody, the hinge region, the transmembrane region, the intracellular co-stimulatory signaling domain, and the intracellular signaling domain, may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

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

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

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is set forth in SEQ ID NO. 14.

Also included herein are nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor or a polynucleotide sequence encoding the PD-1 antibody described herein, and one or more regulatory sequences operably linked to these sequences. In certain embodiments, the nucleic acid construct is an expression cassette.

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

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

In certain embodiments, the nucleic acid construct is a vector. In particular, the coding sequence of the CAR or the coding sequence of the PD-1 antibody herein can be cloned into many types of vectors, for example, such types of vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector may be an expression vector. The expression vector may be provided to the cell as a viral vector. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, in certain embodiments, the invention uses a retroviral vector comprising a replication initiation site, a 3'LTR, a 5' LTR, the coding sequences for CARs described herein or the coding sequences for PD-1 antibodies, and optionally a selectable marker.

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

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

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

In certain embodiments, the coding sequences for the chimeric antigen receptor and the coding sequences for the PD-1 antibody described herein are separately cloned into vectors (also referred to as integration vectors), particularly transposon vectors, for integration of the nucleic acid sequence of interest into the genome of the host cell. In certain embodiments, the transposon vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleep reliability, frog priority, tn5, or Ty. Such transposon vectors contain the 5 'inverted terminal repeat (5' LTR) of the corresponding transposon and the 3 'inverted terminal repeat (3' LTR) of the corresponding transposon. The transposase may be a transposase from a piggybac, sleep bearing, frog priority, tn5 or Ty transposase system. When transposases from different transposition systems are used, the sequences of the 5'LTR and 3' LTR in the vector are also changed accordingly to sequences that fit the transposition system, as can be readily determined by one skilled in the art. Between the 5'ltr and the 3' ltr is an expression cassette for a CAR or antibody of the invention, comprising a corresponding promoter sequence, a coding sequence for the CAR or antibody, and a polyA tailing signal sequence.

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

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

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

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

In certain embodiments, a T cell modified by a Muc1CAR gene and capable of expressing a PD-1 antibody as described herein can be transformed into:

(1) A vector comprising a transposase coding sequence for integration into the T cell genome of the chimeric antigen receptor expression cassette, and a vector comprising a transposase coding sequence for integration into the T cell genome of the expression cassette of the PD-1 antibodies described herein;

(2) A transposase-free vector for integration into the T cell genome of the chimeric antigen receptor expression cassette, and a transposase-free vector for integration into the T cell genome of the expression cassette of the PD-1 antibodies described herein;

(3) A vector comprising a transposase coding sequence for integration into the T cell genome of the chimeric antigen receptor expression cassette, and a vector comprising no transposase coding sequence for integration into the T cell genome of the expression cassette of the PD-1 antibodies described herein; or (b)

(4) A transposase-free coding sequence for integration into the chimeric antigen receptor expression cassette in the T cell genome, and a transposase-containing coding sequence for integration into the expression cassette of a PD-1 antibody described herein in the T cell genome.

Preferably, the T cells are transformed with a vector comprising a transposase coding sequence for integration into the expression cassette of a chimeric antigen receptor in the T cell genome and a vector not comprising a transposase coding sequence for integration into the expression cassette of a PD-1 antibody described herein in the T cell genome. More preferably, the T cells are transformed with a vector comprising a chimeric antigen receptor coding sequence constructed with the pNB328 vector as a backbone vector and a vector comprising a PD-1 antibody expression cassette constructed with the pS328 vector (without a transposase coding sequence as compared to pNB 328) as a backbone vector. In certain embodiments, the chimeric antigen receptor has a coding sequence as set forth in SEQ ID NO. 14; the coding sequence of the PD-1 antibody is shown as the 61 st to 1488 th base sequence of SEQ ID NO. 2. In certain embodiments, the signal peptide of the PD-1 antibody is a light chain signal peptide in the vector comprising the coding sequence of the PD-1 antibody. The amino acid sequence of an exemplary light chain signal peptide may be as shown in amino acid residues 1-20 of SEQ ID NO. 1. More specifically, in certain embodiments, the transposase coding sequence-containing vector having a chimeric antigen receptor coding sequence integrated into the T cell genome comprises, in order, a 5'ltr, a promoter, a CD8 signal peptide coding sequence, a coding sequence for scFv that recognizes Muc1 antigen, a coding sequence for the hinge region of IgG4Fc CH2CH3, a coding sequence for the CD28 transmembrane region, a coding sequence for the CD28 intracellular domain, a coding sequence for the CD3 zeta intracellular signal domain, a polyA tailing signal sequence, a coding sequence for 3' ltr and transposase, and promoters thereof; the vector without transposase coding sequence, which incorporates the coding sequence of the PD-1 antibodies described herein in the T cell genome, contains a promoter, a coding sequence for a light chain signal peptide, a coding sequence for the PD-1 antibody, and a polyA tailing signal sequence in that order between the 5'LTR and the 3' LTR.

Preferably, the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the PD-1 antibody coding sequence is 1-7 during transfection: 1 to 7, preferably 1 to 3:1 to 3, preferably 1:1 to 3, more preferably 1:1 to 2, more preferably 1:1.

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

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

The invention also provides a composition comprising a vector comprising the chimeric antigen receptor expression cassette described herein and a vector comprising the expression cassette of the PD-1 antibody described herein. Suitable agents may also be included in the composition, including but not limited to agents for transfection.

The invention also provides a kit comprising a vector comprising the chimeric antigen receptor expression cassette described herein and a vector comprising the expression cassette of the PD-1 antibody described herein, or a composition described herein. The kit may also be provided with reagents or instruments for transferring the vector into cells.

It is to be understood that the expression cassettes described herein contain at least a suitable promoter and polyA tailing signal sequence in addition to the coding sequences for the CARs or antibodies described herein.

The invention also provides a pharmaceutical composition comprising a T cell as described herein or a PD-1 antibody expressed by the T cell. The pharmaceutical composition may contain suitable pharmaceutically acceptable carriers or excipients. The pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells. The therapeutically or prophylactically effective amount of T cells can be determined based on factors such as the patient's condition.

The invention also provides the use of the T cells or the pharmaceutical composition thereof or the T cells and the PD-1 antibodies expressed by the T cells in the preparation of medicaments for treating or preventing malignant tumors. The invention also provides a method of treating or preventing a malignancy, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the invention. Cancers suitable for treatment or prophylaxis of T cells described herein are preferably Muc1 positive cancers, and specifically include cancers in which Muc1 is abnormally expressed on the surface of cancer cells, such as cancers in which the expression level of Muc1 on the surface of cancer cells is 100 times or more than normal, and in which Muc1 is uniformly distributed on the whole cell surface. In particular, such cancers may be selected from: adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer, or prostate cancer.

Embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, J. Sam. Brookfield et al., huang Peitang et al. Ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Furthermore, it is to be understood that the use of "comprising" herein also includes "consisting of … …".

Example 1: recombinant plasmid pNB328-Muc1CAR, pS328-Muc1CAR, pNB328-m279V, pS- Construction of m279V-wt, pS328-m279V, pNB328-Muc1CAR-2A-m279V, pNB328-m279V-IRES-Muc1CAR And the acquisition of multiple types of pluripotent T cells.

1. Recombinant plasmid construction

The Muc1CAR foreign gene (containing the CD8 signal peptide, antigen recognition single chain antibody, igG4CH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular domain and CD3 zeta tyrosine activation motif) was synthesized by Shanghai JieR biological company, the nucleotide sequence of which is shown in SEQ ID NO:14, the encoded amino acid sequence of which is shown in SEQ ID NO:13, was introduced upstream thereof with a polyclonal restriction site (BglII-XbaI-EcoRI-BamHI), downstream thereof with a restriction site (SalI-NheI-HindIII-SpeI) inserted, and was loaded into pNB328 vector or pS328 vector (see CN 201510638974.7 for the structure and sequence of pNB328, the entire contents of which are incorporated herein by reference; pS328 lacks PB transposon sequence compared to pNB 328) to construct recombinant plasmids designated pNB328-Muc1CAR and pS328-Muc1, respectively.

The foreign gene of wild type PD-1 antibody (comprising the coding sequence of light chain signal peptide, the coding sequence of light chain signal peptide is shown as the 1 st to 60 th base sequence of SEQ ID NO:16, the coding sequence of wild type PD-1 antibody is shown as the 61 st to 1488 th base sequence of SEQ ID NO:16, the amino acid sequence of light chain signal peptide is shown as the 1 st to 20 th amino acid residues of SEQ ID NO: 15), the amino acid sequence of wild type PD-1 antibody is shown as the 21 st to 495 th amino acid residues of SEQ ID NO: 15), the foreign gene of mutant PD-1 antibody (comprising the coding sequence of light chain signal peptide, the coding sequence of light chain signal peptide is shown as the 1 st to 60 th base sequence of SEQ ID NO:2, the coding sequence of mutant PD-1 antibody is shown as the 61 st to 1488 th base sequence of SEQ ID NO:2, the amino acid sequence of the mutant PD-1 antibody is shown as the 1 st to 20 th amino acid residues of SEQ ID NO:15 th amino acid residues, the amino acid sequence of mutant PD-1 is shown as the 1 st to 495 th amino acid residues of SEQ ID NO: 21 st to 495, and the mutant PD-1 is inserted into the plasmid pS I-328 to be a multiple position of the plasmid (328 to 328 m), and the plasmid is inserted into the plasmid (328) of the plasmid by the double-328 to form the recombinant vector by the double-restriction enzyme, respectively, the plasmid is inserted into the plasmid (328-328, and the plasmid is inserted into the plasmid).

The aforementioned synthetic Muc1CAR exogenous gene was ligated with the exogenous gene of the mutant PD-1 antibody in 2A (SEQ ID NO:17, amino acid sequence shown as SEQ ID NO: 18), and a polyclonal cleavage site (BglII-XbaI-EcoRI-BamHI) was introduced upstream thereof, and a cleavage site (SalI-NheI-HindIII-SpeI) was inserted downstream thereof, and this was placed into the pNB328 vector double digested with EcoR1+SalI to construct a recombinant plasmid designated pNB328-Muc1CAR-2A-m279V.

The foreign gene of the aforementioned synthesized mutant PD-1 antibody was ligated with the foreign gene of Muc1CAR by IRES (SEQ ID NO: 19), and a polyclonal cleavage site (BglII-XbaI-EcoRI-BamHI) was introduced upstream thereof, and a cleavage site (SalI-NheI-HindIII-SpeI) was inserted downstream thereof, and this was placed into the pNB328 vector double-digested with EcoR1+SalI to construct a recombinant plasmid designated pNB328-m279V-IRES-Muc1CAR.

The structure of each recombinant plasmid constructed is shown in FIG. 1. The promoter sequence and polyA tailing signal sequence are not shown in the structural schematic diagrams, and are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.

2. Obtaining Muc1CAR-anti PD1 pluripotent T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from donor blood using Filcoll separation. Culturing PBMC for 2-4h in an adherence way, wherein non-adherence suspension cells are initial T cells, collecting the suspension cells into a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps;

For each recombinant plasmid 6 1.5ml centrifuge tubes were taken, numbered a, b, c, d, e, f and g. 5X 10 of each tube was added ⁶ The individual cells were centrifuged at 1200rmp for 3min, the supernatant was discarded, an electrotransfer kit (from Lonza Corp.) was taken, 100ul of electrotransfer reagent was added per tube, 4ug recombinant plasmid pS328-Muc1CAR and 4ug recombinant plasmid pNB328-m279V (mass ratio 1:1) were added to tube a, 4ug recombinant plasmid pNB328-Muc1CAR and 4ug recombinant plasmid pS328-m279V (mass ratio 1:1) were added to tube b, 8ug pNB328-Muc1CAR-2A-m279V plasmid was added to tube c, 8ug pNB328-m279V-IRES-Muc1CAR was added to tube d, 8ug pNB328 vector plasmid was added to tube f, 4ug recombinant plasmid pNB328-Muc1CAR and 4ug recombinant plasmid pNB328-Muc1CAR were added to tube f, 4ug recombinant plasmid pNB328-Muc1 was added to tube g, and 4ug recombinant plasmid pNB328-Muc1 was resuspended, respectively, and the cells were resuspended; transferring the mixed solution to an electric rotating cup, putting the electric rotating cup into an electric rotating instrument, selecting a required program, and performing electric shock; another 4 1.5ml centrifuge tubes, numbered 1,2,3,4, were likewise taken, 5X 10 per tube ⁶ The individual cells were centrifuged at 1200rmp for 3min, the supernatant was discarded, an electrotransfer kit (from Lonza Corp.) was taken, 100ul of electrotransfer reagent was added per tube in proportion, recombinant plasmid pNB328-Muc1CAR and recombinant plasmid pS328-m279V were added per tube in different mass ratios (1:1, 3:5, 1:3, 1:7), the electrotransfer cell suspension was transferred to six well plates (AIM-V culture solution containing 2% FBS) in the kit, and mixed, placed in a 37℃5% CO2 incubator for culture, and after 6 hours, the stimulating factors IL-2 and Muc1 antigen, CD28 antibody were added, and after 3-4 days of culture at 37℃5% CO2, the growth of T cells were observed to obtain corresponding T cells, named pS328-Muc1 +pNB 328-anti-iPD 1T cells, pNB328-Muc1 +pS328-anti 1T cells, muc1 +pS 328-anti-iPr 1T cells, muc 1-iPr 1T cells, and pS 1-iPr 1-3-iPr 1T cells, respectively, and Muc 1-iPr 1-3-iPr 1T cells were obtained.

Example 2: PBMCs via the Muc1CAR geneModified and activated expression of PD-1 antibody genes in different combination forms And (3) determining the positive rate of the Muc1CAR gene and the expression of the PD-1 antibody.

The above constructed activated T cells of different kinds and different patient sources were cultured according to 1X 10 on day 12 after electrotransformation ⁶ Cell pellet was washed 2 times with PBS, added with 2.5ul of Biotin-Muc1 antibody, incubated at 4℃for 30min, washed 2 times with PBS, added with 2ul of PE-streptomycin secondary antibody, washed 2 times with PBS, transferred to flow tubes with 400ul of PBS, and checked on-machine. Likewise, 2X 10 is collected ⁶ Cell number and number of cells was 2X 10 ⁶ Cells/well were plated in 6-well plates with 3ml AIM-V culture medium, incubated in a 5% CO2 incubator at 37℃and the cell supernatants were collected after 24h of incubation and stored at-20℃for further use. The expression level of PD-1 antibodies in the genetically modified T cells is quantitatively detected by a double-antibody sandwich ELISA method (detection by using a human PD-1 recombinant protein coated ELISA plate and an HRP-labeled mouse anti-human IgG4mAb, and taking a commercial PD-1 antibody as a standard substance, wherein a sample to be detected is diluted by 50 times.

Results as shown in figures 2A and 2B and the following table, T cells obtained by electrotransformation of the pNB328-Muc1CAR plasmid and pS328-m279V plasmid combination have higher Muc1CAR gene and PD-1 antibody secretion:

T cell type	PD-1 antibody expression level (ng/ml)	CAR-T cell positive rate (%)
			Mock T	0.466	0.31
pS328-Muc1CAR+pNB328-antiPD1T	1590.49	45.93
			pNB328-Muc1CAR+pS328-antiPD1T	2190.49	91.17
Muc1CAR-2A-antiPD1T	105.20	10.38
			antiPD1-IRES-Muc1CAR T	366.47	35.33

Example 3: PBMCs were modified for expression by different mass ratios of pNB328-Muc1CAR to pS328-m279V plasmid Muc1CAR gene positive rate and PD-1 antibody expression amount determination

The recombinant plasmids pNB328-Muc1CAR and pS328-m279V were electrotransformed into PBMCs cells (1:1, 3:5, 1:3, 1:7) at different mass ratios as described in example 1 to obtain a variety of T cells simultaneously expressing the Muc1CAR gene and the PD-1 antibody. These T cells were plated at 2X 10 on day 12 of post-electrotransformation culture ⁶ Cell number cells were collected and grown at 2X 10 ⁶ Cells/well were plated in 6-well plates with 3ml AIM-V culture medium, incubated in a 5% CO2 incubator at 37℃and the cell supernatants were collected after 24h of incubation and stored at-20℃for further use. By a double-antibody sandwich ELISA method (detection by using human PD-1 recombinant protein coated ELISA plate and HRP-labeled mouse anti-human IgG4mAb, detection by using commercial PD-1 antibody as a standard, quantitative detection of the expression level of the PD-1 antibody in the genetically modified T cells after 50-fold dilution of a sample to be detected, simultaneous collection of 1×10) ⁶ Cell pellet, washed 2 times with PBS, and added 2.5ulBiotin-Muc1 antibody, incubation at 4deg.C for 30min, PBS wash cell pellet 2 times, adding 2ul PE-streptomycin secondary antibody, PBS wash cell pellet 2 times, adding 400ul PBS transfer cells into flow tube, and on-machine detection.

As a result, it was found that the pNB328-Muc1CAR plasmid and the pS328-m279V plasmid were used as a vector 1 as shown in FIGS. 3A, 3B and the following tables: the T cells obtained by electrotransformation have higher Muc1CAR genes and PD-1 antibody secretion.

Example 4: t cells of PBMCs from different patients after modification and activation of Muc1CAR gene and PD-1 antibody gene Determination of the positive rate of the gene expressing Muc1 CAR.

Mock T cells, muc1CAR T cells, and Muc1 CAR-anti-ipd 1T cells transformed with plasmids pNB328-Muc1CAR and pS328-m279V obtained in example 1 were cultured for different patients at 1×10 on day 12 ⁶ Cell pellet was collected, washed 2 times with PBS, added with 2.5ul of Biotin-Muc1 antibody, incubated at 4℃for 30min, washed 2 times with PBS, added with 2ul of PE-streptomycin secondary antibody, washed 2 times with PBS, transferred to a flow tube with 400ul of PBS, and checked on-machine.

The results are shown in FIG. 4, where the Muc1CAR recombinant protein can be stably expressed on the surface of T cells.

Example 5: t cells of pBMCs from different patients after modification and activation of Muc1CAR gene and PD-1 antibody gene And (3) quantitatively detecting the expression quantity of the expressed PD-1 antibody.

Mock T cells, muc1CAR T cells and Muc1 CAR-anti-ipd 1T cells transformed with plasmids pNB328-Muc1CAR and pS328-m279V obtained in example 1 were cultured for different patients at day 12 at 2×10 ⁶ Cell number cells were collected and grown at 2X 10 ⁶ Cells/well were plated in 6-well plates with 3ml AIM-V culture medium, incubated in a 5% CO2 incubator at 37℃and the cell supernatants were collected after 24h of incubation and stored at-20℃for further use. By two pairsAntibody sandwich ELISA method (detection by using human PD-1 recombinant protein coated ELISA plate and HRP-labeled mouse anti-human IgG4mAb, and quantitative detection of PD-1 antibody expression in T cell modified by Muc1CAR gene and PD-1 antibody gene after 50-fold dilution of sample to be detected.

The results are shown in figure 5, where Muc1CAR T cells genetically modified with PD-1 antibodies were able to stably express PD-1 antibodies at high levels.

Example 6: t cells of PBMCs from different patients modified by Muc1CAR gene and PD-1 antibody gene Detection of Muc1CAR genome expression levels in the genome.

Genomic DNAs of Mock T cells, muc1CAR T cells and Muc1 CAR-anti-iPD 1T cells transformed with plasmids pNB328-Muc1CAR and pS328-m279V obtained in example 1 were extracted (kit method), DNA concentrations of the Mock T cells, muc1CAR T cells and Muc1 CAR-anti-iPD 1T cells were measured by referring to instructions attached to the kit in the experimental procedure, and the expression level of the Muc1CAR genome was detected by using a real-time fluorescent quantitative PCR method, and the reaction procedure was as follows: 50 ℃,2 min-95 ℃,10 min-95 ℃,15 s-60 ℃,1min,40 cycles. And calculating the absolute copy number content according to a corresponding formula by using the CT value of the obtained Muc1CAR genome and the CT value of the action.

As a result, it was found that the Muc1CAR genome was integrated into the T cell genome via the PB transposase system, as shown in the following table:

example 7: flow detection of Mock T cells, muc1CAR T cells and Muc1CAR-antiPD1T cell activation tables Differentiation between type and cytokine secretion.

1. Collecting the suspended Mock T cells obtained in example 1, and mu c1CAR T cells and mu c1 CAR-anti-iPD 1T cells transferred with pNB328-mu c1CAR and pS328-m279V plasmids, washing twice with PBS, centrifuging at 1200rpm for 5min, and adding 2ul of isotype control antibody IgG1-PE, fluorescent flow antibody anti-CD25-PE, anti-LAG3-PE and anti-PD1-PE respectively; isotype control antibody IgG1-PC5, fluorescent flow antibody anti-CD107 α -PC5; isotype control antibody IgG1FITC, fluorescent flow-through antibody anti-CD62L-FITC; isotype control antibody IgG1-PC5, fluorescent flow antibody anti-CD45RO-PC5; the isotype control antibody IgG1-PE, the fluorescent flow antibody anti-CCR7-PE and the flick precipitation are uniformly mixed, incubated for 30min at room temperature and in a dark place, washed once by PBS, and 400ul of PBS is added to transfer cells into a flow tube for detection on the machine.

The result shows that the PD-1 single-chain antibody secreted by the Muc1 CAR-anti-iPD 1T cell can well seal PD-1 protein on the surface of the T cell, the Muc1CAR T cell and the Muc1 CAR-anti-iPD 1T cell have obvious killing activity in vitro, the formation of effector memory T can be promoted, the activation mark CD25 is obviously higher than that of the Mock T cell, and the depletion mark LAG3 of the Muc1 CAR-anti-iPD 1T cell is obviously lower than that of the Mock T cell and the Muc1CAR T cell, and the specific is shown in figures 6A and 6B.

2. Collection of 1X 10 ⁶ Muc1CAR T cells obtained in example 1 and Muc1 CAR-anti-iPD 1T cells transformed with pNB328-Muc1CAR and pS328-m279V plasmids were added to a 1.5ml EP tube, respectively, washed twice with PBS, centrifuged at 1200rpm for 5min, added with 2ul of alpha-CD 3CD4CD8 antibody, incubated at room temperature for 30min in the absence of light, washed once with PBS, transferred to a flow tube with 400ul of PBS, and detected on-line.

As a result, it was found that CD3 in Muc1CAR T cells, muc1CAR-anti PD1T cells and Mock T ⁺ CD4 ⁺ ，CD3 ⁺ CD8 ⁺ The percentage of cells was not greatly different, as shown in particular in fig. 6C.

3. Coating 24-well plate with 5ug/ml Muc1 antigen, coating overnight at 4deg.C, washing 3 times with PBS, adding 3×10 ⁵ Mock T cells obtained in example 1, muc1CAR T cells and Muc1CAR-anti pd1T cells transformed with plasmids pNB328-Muc1CAR and pS328-m279V were cultured for 24 hours and cell supernatants were collected. By BD ^TM CBA Human Th1/Th2Cytokine Kit II detection of secretion of cytokines from Muc1CAR T cells and Muc1 CAR-anti-iPD 1T cells stimulated by Muc1 antigen:

(1) Mixing human IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma capturing magnetic beads, vortex oscillating and mixing the capturing magnetic beads, and adding 50ul of the uniformly mixed capturing magnetic beads into each tube;

(2) 50ul of human Th1/Th2cytokine standard (dilution of power ratio 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml, 20pg/ml, 0 pg/ml) and 50ul of sample to be tested (2-fold dilution with diluent) were added;

(3) 50ul of human Th1/Th2-II-PE detection antibody was added to each tube;

(4) Incubating for 3 hours at room temperature in a dark place;

(5) 1ml of washing buffer solution is added into each tube, 200g is centrifuged for 5min, and the supernatant is discarded;

(6) Cells were resuspended by adding 300ul of wash buffer per tube and transferred to flow tubes and fluorescence values were detected by flow cytometry.

As a result, it was found that Muc1CAR T cells and Muc1 CAR-anti-iPD 1T cells secreted IL-2, TNF- α and IFN- γ, which were significantly improved over Mock T cells, while the three cells secreted IL-4, IL-6 and IL-10, which were not different, as shown in FIG. 6D.

Example 8: mock T cells, muc1CAR T cells, and Muc1CAR-anti pd1T cells were pair cultured in vitro Killing experiments of tumor cells.

Selecting effector cells and target cells which are matched with MHC class I in a typing way, and detecting the in-vitro killing activity of the cells by using a real-time label-free cell function analyzer (RTCA), wherein the method comprises the following specific steps of:

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

(2) Target cell plating: cervical cancer cell Hela, liver cancer cell HCC-LM3, lung cancer cell A549 (purchased from American type culture Collection ATCC) at 10 per well ⁴ Spreading the individual cells/50 μl in a plate containing detection electrodes, standing for several minutes, placing into an instrument after the cells are stabilized, and starting step 2 to culture the cells;

(3) Adding effector cells: after the target cells were cultured for 24 hours, step 2 was suspended, effector cells (Mock T cells obtained in example 1, muc1CAR T cells and Muc1CAR-anti pid 1T cells transformed with plasmids pNB328-Muc1CAR and pS328-m 279V) were added, 50 μl per well was used, the target ratio was set to 4:1, and step 3 was started with Mock T cells not transformed with plasmids as a control, and the co-culture was continued for 24 hours to obtain a cell proliferation curve.

The results show that Muc1CAR T cells expressing PD-1 antibodies kill all three tumor cells better than Muc1CAR T cells. As particularly shown in fig. 7.

Example 9: in vivo functional studies of Muc1CAR T cells expressing PD-1 antibodies.

The first step: 15 NSG complete immunodeficiency mice of 4-6 weeks old, average weight 22-27 g, were raised by SPF grade animal laboratory supplied by Beijing Viway Biotechnology Co.

And a second step of: culturing human cervical cancer cells Hela in vitro, taking adherent growth cells in logarithmic growth phase, digesting with 0.25% pancreatin, centrifuging, collecting cells, re-suspending with PBS solution, centrifuging at 3000g room temperature for 2 min, discarding supernatant, re-suspending with PBS solution, centrifuging, collecting cells, and adjusting cell suspension concentration to 5×10 ⁷ And each ml.

And a third step of: the back of the right rib of the mouse was inoculated subcutaneously with Hela-luc cells, 0.1 ml/mouse. Tumor size was observed by a biopsy imager after about 10 days of inoculation, NSG immunodeficient mice were randomly divided into 4 groups of 5 mice each, and PBS (100 ul) was administered to each group, and the Mock T cells, muc1CAR-antiPD1-wt T cells transformed with pNB328-Muc1CAR and pS328-m279V-wt, and Muc1CAR-antiPD1T cells transformed with pNB328-Muc1CAR and pS328-m279V plasmids (1X 10) ⁷ And/or just). The administration route is tail vein injection.

Fourth step: mice were observed daily for their state of life and every 10 days for tumor changes by a biopsy imager.

The results are shown in FIG. 8.

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

Sequence listing

<110> Shanghai cell therapy institute

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

<120> muc 1-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof

<130> 17A007

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

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

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly

35 40 45

Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

100 105 110

Gln His Ser Arg Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Asn Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

180 185 190

Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu

195 200 205

Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr

210 215 220

Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp

245 250 255

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

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 2

<211> 1488

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180

caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240

ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300

agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360

acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420

agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480

ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540

tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600

aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660

tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720

tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780

accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840

gagttcgagg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt tccagagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488

<210> 3

<211> 228

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

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

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly Lys

225

<210> 4

<211> 684

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gagtccaaat atggtccccc atgcccacca tgcccagcac ctcccgtggc cggaccatca 60

gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 180

gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt ccagagcacg 240

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc 360

aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 540

tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 600

gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 660

agcctctccc tgtctctggg taaa 684

<210> 5

<211> 22

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

His Ala Ala Arg Pro Ser

20

<210> 6

<211> 45

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys

1 5 10 15

Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val

20 25 30

Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala

35 40 45

<210> 7

<211> 247

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

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

130 135 140

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr Ala Met

145 150 155 160

Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr

165 170 175

Ile Ser Ser Gly Gly Thr Tyr Ile Tyr Tyr Pro Asp Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln

195 200 205

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

210 215 220

Leu Gly Gly Asp Asn Tyr Tyr Glu Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235 240

Thr Thr Val Thr Val Ser Ser

245

<210> 8

<211> 55

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

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

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp

50 55

<210> 9

<211> 28

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 10

<211> 41

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 11

<211> 42

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 12

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 13

<211> 678

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

His Ala Ala Arg Pro Ser Asp Ile Val Ile Thr Gln Ser Thr Ala Ser

20 25 30

Leu Gly Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser

35 40 45

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

50 55 60

Arg Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu

65 70 75 80

Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr

100 105 110

Tyr Cys Gln His Ser Arg Glu Leu Pro Phe Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

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

165 170 175

Phe Ser Gly Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg

180 185 190

Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Thr Tyr Ile Tyr Tyr

195 200 205

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

210 215 220

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

225 230 235 240

Met Tyr Tyr Cys Ala Arg Leu Gly Gly Asp Asn Tyr Tyr Glu Tyr Phe

245 250 255

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys

260 265 270

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Gln Ala Leu Pro Pro Arg

675

<210> 14

<211> 2037

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgaca ttgtgatcac acagtctaca gcttccttag gtgtatctct ggggcagagg 120

gccaccatct catgcagggc cagcaaaagt gtcagtacat ctggctatag ttatatgcac 180

tggtaccaac agagaccagg acagccaccc aaactcctca tctatcttgc atccaaccta 240

gaatctgggg tccctgccag gttcagtggc agtgggtctg ggacagactt caccctcaac 300

atccatcctg tggaggagga ggatgctgca acctattact gtcagcacag tagggagctt 360

ccgttcacgt tcggaggggg gaccaagctg gagataaaag gtggaggcgg ttcaggcgga 420

ggtggcagcg gcggtggcgg gtcggaggtc cagctggagg agtcaggggg aggcttagtg 480

aagcctggag ggtccctgaa actctcctgt gcagcctctg gattcacttt cagtggctat 540

gccatgtctt gggttcgcca gactccggag aagaggctgg agtgggtcgc aaccattagt 600

agtggtggta cttatatcta ctatccagac agtgtgaagg ggcgattcac catctccaga 660

gacaatgcca agaacaccct gtacctgcaa atgagcagtc tgaggtctga ggacacggcc 720

atgtattact gtgcaagact tgggggggat aattactacg aatacttcga tgtctggggc 780

gcagggacca cggtcaccgt ctcctccgag tccaaatatg gtcccccatg cccaccatgc 840

ccagcacctc ccgtggccgg accatcagtc ttcctgttcc ccccaaaacc caaggacact 900

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 960

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 1020

ccgcgggagg agcagttcca gagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1080

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 1140

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 1200

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1260

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1320

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta 1380

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 1440

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa acccttttgg 1500

gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt 1560

attattttct gggtgaggag taagaggagc aggctcctgc acagtgacta catgaacatg 1620

actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 1680

ttcgcagcct atcgctccag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1740

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1800

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1860

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1920

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1980

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctga 2037

<210> 15

<211> 495

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly

35 40 45

Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

100 105 110

Gln His Ser Arg Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Asn Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

180 185 190

Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu

195 200 205

Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr

210 215 220

Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 16

<211> 1488

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120

ctctcctgca gggccagcaa aggtgtcagt acatctggct atagttattt gcactggtat 180

caacagaaac ctggccaggc tcccaggctc ctcatctatc ttgcatccta cctagaatct 240

ggcgtcccag ccaggttcag tggtagtggg tctgggacag acttcactct caccatcagc 300

agcctagagc ctgaagattt tgcagtttat tactgtcagc acagcaggga ccttccgctc 360

acgttcggcg gagggaccaa agtggagatc aaaggtggag gcggttcagg cggaggtggc 420

agcggcggtg gcgggtcgca ggtgcagctg gtgcagtccg gcgtggaggt gaagaagcct 480

ggcgcctccg tcaaggtgtc ctgtaaggcc tccggctaca ccttcaccaa ctactacatg 540

tactgggtgc ggcaggcccc aggccaggga ctggagtgga tgggcggcat caacccttcc 600

aacggcggca ccaacttcaa cgagaagttc aagaaccggg tgaccctgac caccgactcc 660

tccaccacaa ccgcctacat ggaactgaag tccctgcagt tcgacgacac cgccgtgtac 720

tactgcgcca ggcgggacta ccggttcgac atgggcttcg actactgggg ccagggcacc 780

accgtgaccg tgtcctccga gtccaaatat ggtcccccat gcccaccatg cccagcacct 840

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 900

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 960

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1200

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1260

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1380

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatga 1488

<210> 17

<211> 78

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

cgtaggaaac gaggcagcgg cgccacaaac ttctctctgc taaagcaagc aggtgatgtt 60

gaagaaaacc ccgggcct 78

<210> 18

<211> 26

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Arg Arg Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln

1 5 10 15

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 19

<211> 197

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ccggcgggtt tctgacatcc ggcgggtttc tgacatccgg cgggtttctg acatccggcg 60

ggtttctgac atccggcggg tttctgacat ccggcgggtt tctgacatcc ggcgggtttc 120

tgacatccgg cgggtttctg acatccggcg ggtttctgac atccggcggg tgactcacaa 180

ccccagaaac agacata 197

Claims

1. A T cell, wherein the T cell:

(1) Comprising a coding sequence for expressing a chimeric antigen receptor that recognizes the Muc1 antigen and a coding sequence for a PD-1 antibody; and/or

(2) Expressing chimeric antigen receptor recognizing Muc1 antigen and PD-1 antibody,

the PD-1 antibody comprises an anti-PD-1 single-chain antibody and IgG4Fc; wherein the amino acid sequence of the IgG4Fc is shown as amino acid residues 267-495 of SEQ ID NO. 1, the amino acid sequence of the light chain variable region of the anti-PD-1 single-chain antibody is shown as amino acid residues 21-131 of SEQ ID NO. 1, the amino acid sequence of the heavy chain variable region of the anti-PD-1 single-chain antibody is shown as amino acid sequences 147-266 of SEQ ID NO. 1,

the chimeric antigen receptor sequentially comprises a signal peptide, an anti-Muc 1 near-membrane single-chain antibody, an IgG4Fc CH2CH3 hinge region, a CD28 transmembrane region, a CD28 intracellular domain and a tyrosine activation motif of CD3 zeta from the N end to the C end, wherein the amino acid sequence of the Muc1 near-membrane single-chain antibody is shown as SEQ ID NO. 7.

2. The T cell of claim 1, wherein the T cell has integrated into its genome an expression cassette for a chimeric antigen receptor that recognizes the Muc1 antigen and an expression cassette for a PD-1 antibody.

3. The T cell of claim 1, wherein the signal peptide is a CD8 signal peptide.

4. The T cell of claim 3, wherein the amino acid sequence of the CD8 signal peptide is set forth in SEQ ID NO. 5.

5. The T cell of claim 1, wherein the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is set forth in SEQ ID No. 9.

6. The T cell of claim 1, wherein the amino acid sequence of the CD28 transmembrane region is set forth in SEQ ID No. 9.

7. The T cell of claim 1, wherein the intracellular domain of CD28 has the amino acid sequence shown in SEQ ID No. 10.

8. The T cell of claim 1, wherein the amino acid sequence of the cd3ζ intracellular signaling domain is depicted as SEQ ID No. 12.

9. The T cell of any one of claims 1-8, wherein the chimeric antigen receptor has one or more of the following characteristics:

the coding sequence of the signal peptide is shown as 1 st to 66 th base of SEQ ID NO. 14;

The coding sequence of the single-chain antibody is shown as 67 th to 807 th bases of SEQ ID NO. 14;

the coding sequence of the hinge region is shown in 808-1491 of SEQ ID NO. 14;

the coding sequence of the transmembrane region is shown as 1492-1575 bases of SEQ ID NO. 14;

the coding sequence of the intracellular co-stimulatory signal domain is shown as the 1576 th to 1698 th bases of SEQ ID NO. 14; and

the coding sequence of the intracellular signal domain is shown as 1699 th to 2034 th bases of SEQ ID NO. 10.

10. The T cell of claim 1, wherein the chimeric antigen receptor has an amino acid sequence as set forth in amino acid residues 23-678 of SEQ ID No. 13.

11. The T cell of claim 10, wherein the chimeric antigen receptor has a coding sequence as set forth in bases 70-2034 of SEQ ID No. 14.

12. The T cell of claim 1, wherein the PD-1 antibody further comprises a light chain signal peptide.

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

14. The T cell of claim 1, wherein the anti-PD-1 single chain antibody has an amino acid sequence as set forth in amino acid residues 21-266 of SEQ ID No. 1, or as set forth in SEQ ID No. 1.

15. The T cell of claim 14, wherein the anti-PD-1 single chain antibody has a coding sequence as set forth in SEQ ID No. 2 at positions 61-798 or as set forth in SEQ ID No. 2.

16. The T cell of claim 14, wherein the PD-1 antibody has an amino acid sequence as set forth in SEQ ID No. 1 at amino acid positions 21-495 or as set forth in SEQ ID No. 1.

17. The T cell of claim 16, wherein the PD-1 antibody has a coding sequence as set forth in amino acid residues 61-1485 of SEQ ID No. 2 or as set forth in SEQ ID No. 2.

18. A composition or kit comprising:

(1) A vector comprising an expression cassette for a chimeric antigen receptor as defined in any one of claims 1 to 11 for integration of said expression cassette into the genome of a host cell; and

(2) A vector comprising an expression cassette for a PD-1 antibody as defined in any one of claims 12-17 for integration of the expression cassette into the genome of a host cell.

19. A pharmaceutical composition comprising the T cell of any one of claims 1-17 or the T cell and its expressed PD-1 antibody.

20. Use of a T cell according to any one of claims 1 to 17 or of said T cell and its expressed PD-1 antibody in the manufacture of a medicament for the treatment or prophylaxis of cancer, wherein said cancer is a cancer whose cancer cell surface abnormally expresses Muc 1.

21. The use according to claim 20, wherein the cancer is a cancer in which the expression level of Muc1 on the surface of cancer cells is 100 times or more that in normal cases, and wherein Muc1 is uniformly distributed on the whole cell surface.

22. The use of claim 20, wherein the cancer is selected from the group consisting of: lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gall bladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.

23. The use of claim 20, wherein the cancer is an adenocarcinoma.