CN113583139A

CN113583139A - Chimeric receptor and application thereof

Info

Publication number: CN113583139A
Application number: CN202110761462.5A
Authority: CN
Inventors: 徐建青; 张晓燕; 廖启彬; 丁相卿
Original assignee: Shanghai Sinobay Bio Tech Co ltd
Current assignee: Shanghai Sinobay Bio Tech Co ltd
Priority date: 2020-07-06
Filing date: 2021-07-06
Publication date: 2021-11-02
Also published as: WO2022007795A1

Abstract

The present invention provides a chimeric receptor comprising: (1) an extracellular recognition domain of an Fc fragment; (2) an extracellular spacer region; (3) a transmembrane region; and (4) a cellular Internal signaling domains; optionally, the chimeric receptor further comprises (5) one or more costimulatory signaling domains and/or (6) one or more cytokine receptor signaling domains. The present invention also provides immune cells expressing the chimeric receptor. The present invention also provides a combination comprising the immune cell and a tumor antigen-targeting antibody or a viral antigen-targeting antibody. The present invention provides a brand-new Fc fragment high-affinity chimeric receptor, which has higher antibody Fc fragment affinity, and can be used in combination with tumor antigen-targeting antibodies or viral antigen-targeting antibodies, which can significantly enhance the effect of ADCC.

Description

Chimeric receptor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines. In particular, the present invention relates to a chimeric receptor. The invention also relates to the chimeric receptor and application thereof.

Background

2013 is an important turning point for cancer treatment, and cancer immunotherapy based on reversing the immunosuppressive microenvironment of body tumor obtains the first ten scientific breakthroughs in 2013 published by science. Subsequently, Chimeric receptor T-cell immunotherapy (CAR-T) showed significant efficacy in the treatment of hematological tumors. CAR-T cell therapy by viral or non-viral exogenous gene delivery technology, a fusion protein that recognizes the recognition domain of a tumor specific/associated antigen and a T cell activation-related sequence is expressed on the surface of a T cell, allowing it to bind specifically to the tumor antigen in an antigen-dependent but MHC-independent manner, initiating activation and specifically killing the tumor cell. However, in solid tumors, the use of this therapy is limited due to the lack of specific tumor antigens.

Most of currently used tumor antigens are tumor-associated antigens, are highly expressed in tumor cells, but are also slightly expressed in normal tissue cells, so that the CAR-T cell therapy has an On-target off-tumor (non-tumor) toxic and side effect, and the clinical application of the CAR-T cell therapy in solid tumor treatment is limited. Results of a CAR-T clinical study targeting Carbonic Anhydrase IX (CAIX) showed that CAIX CAR-T cell therapy not only failed to control patient tumor growth, but instead led to patient liver function abnormalities and the development of cholangitis (Lamers CH, Sleijfer S, Vulto AG, et al.treatment of pathological renal cells with autologous T-lymphocytes genetically specific reached the onset of infection carbonic acid and drug delivery IX: first clinical experiment [ J ]. J Clin Oncol.2006,24(13): e20-e 22). Another CAR-T cell therapy targeting tyrosine kinase receptor 2(ERBB2) is more lethal to the inflammatory factor storm leading to patient death (Morgan RA, Yang JC, Kitano M, et al, case report of a serous uptake effect following the administration of T cells transformed with a molecular inhibitor receptor registering ERBB2[ J ] Mol Ther.2010,18: 843-. Therefore, there is a need to develop a safer immune cell therapy technology.

In addition, clinical studies have found that the recurrence rate following treatment of hematological tumors with CAR-T cell therapy is as high as 50%, and in particular that CD19/CD22 is susceptible to antigen escape following CAR-T cell therapy (Shah NN, Fry TJ. mechanisms of resistance to CAR T cell therapy [ j ]. Nat Rev Clin Oncol.2019,16(6): 372-385). Because the original CAR-T cells targeting the target cannot control tumor growth due to mutation or loss of the antigen of interest, CAR-T cell therapy that requires switching to target a new antigen can inhibit tumor growth. The preparation and quality control of the new target CAR-T cells greatly increase the time cost and economic burden of treatment of patients. Therefore, there is a need to develop a universal immune cell therapy technology, which can flexibly switch different tumor antigen targeting antibodies to achieve the purpose of effectively treating one or more tumors.

The CD16 molecule, also known as Fc γ RIII, is expressed primarily in Natural killer cells (NK), neutrophils, and macrophages. CD16 molecules with killer function cells bind to the Fc fragment of antibodies and thereby initiate Antibody-dependent cell-mediated cytotoxicity (ADCC), in which NK cells are the major contributor to the effect. A growing number of studies have reported that the killing of different tumor cells can be achieved by replacing the extracellular Single-chain antibody (scFv) of a chimeric receptor for recognizing a tumor antigen with the wild-type CD16 molecule by a combination of multiple tumor antigen-targeting antibodies (Cl menceau B, Congy-journal N, Gallot G, et al, antibody-dependent cellular cytotoxicity (ADCC) mediated by genetic modification of anti-tumor-specific human T lymphocytes [ J. blood.2006,107(12): 4669-4677; Ochi F, Fujiwara H, Tanimoto K, et al, Gene-modified human α/β -T cells expressing CD 6332-polypeptide expressed CD 32-polypeptide J. 249: 3. Cancer receptor J. 201432. 9).

The research shows that the CD16 molecule has gene polymorphism, the wild CD16 molecule has low affinity and weakened antibody dependent cell mediated cytotoxicity. Another study showed that disintegrin metalloprotease 17(ADAM17) expressed after immune cell activation or present in the external environment can cleave the CD16 molecule on the surface of cell membrane and down-regulate the expression of CD16 on the surface of cell membrane, resulting in impaired immune cell function, especially NK cell function (Romee R, Foley B, Lenvik T, et al. NK cell CD16surface expression and function is regulated by a ligand and a metalloprotease-17(ADAM17) [ J ]. blood.2013,121(18):3599 3608).

In view of the above, it would be desirable to construct a chimeric receptor that specifically binds to an Fc fragment, particularly a High-affinity, cellular protease-resistant CD16(High-affinity, protease-resistant CD16, hrCD16) chimeric receptor.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a chimeric receptor. The chimeric receptor provided by the invention, particularly the hrCD16 chimeric receptor, has high Fc affinity and cellular protease tolerance, can be introduced into various killer immune cells such as T cells, NK cells or NKT cells and the like by a virus/non-virus delivery mode, and can be stably expressed on the cells. The immune cells modified by the chimeric receptor, particularly the hrCD16 chimeric receptor, can be combined with a plurality of tumor antigen targeting antibodies or virus antigen targeting antibodies to kill different types of tumor cells and viruses in a broad spectrum. Because the chimeric receptor can exert ADCC effect only when combined with the tumor antigen targeted antibody or the virus antigen targeted antibody, and further kill tumor cells or cells infected by viruses, the method provided by the application has the advantages of improving the treatment safety, enhancing the curative effect, avoiding tumor antigen escape or virus escape, quickly and flexibly switching the treatment target antibody and the like.

The invention achieves the purpose through the following technical scheme:

in one aspect, the present invention provides a chimeric receptor comprising:

(1) an extracellular recognition domain of an Fc fragment;

(2) an extracellular spacer region;

(3) a transmembrane region; and

(4) an intracellular signaling domain;

optionally, the chimeric receptor further comprises:

(5) one or more co-stimulatory signaling domains; and/or

(6) One or more cytokine receptor signaling domains;

wherein the extracellular recognition domain of the Fc fragment is a CD16 extracellular domain, a CD32 extracellular domain, a CD64 extracellular domain, a CD89 extracellular domain, a CD23 extracellular domain, a fcsri extracellular domain, an FcRn extracellular domain, an Fc binding antibody, Protein a, Protein G, or a mutant or multiple repeat tandem extracellular domain thereof that specifically binds to an antibody Fc fragment;

preferably, the extracellular recognition domain of the Fc fragment is the CD16 extracellular domain;

preferably, the extracellular recognition domain of the Fc fragment is a wild-type CD16 extracellular domain, a F176V mutant CD16 extracellular domain, a S197P mutant CD16 extracellular domain, or a F176V and S197P double mutant CD16 extracellular domain comprising the amino acid sequence set forth in any one of SEQ ID NOs 1-4; more preferably, the extracellular recognition domain of the Fc fragment is the F176V and S197P double mutant CD16 extracellular domains comprising the amino acid sequence set forth in SEQ ID No. 4.

The chimeric receptor according to the invention, characterized in that the extracellular spacer region includes, but is not limited to, any one or more of the following:

(1) the hinge region of antibody IgG4 and mutants thereof;

(2) the hinge region of antibody IgG4 and mutants thereof and the CH2 region;

(3) the hinge region of antibody IgG4 and its mutants, the CH2 region and the CH3 region;

(4) the hinge region of antibody IgG1 and mutants thereof;

(5) the hinge region of antibody IgG1 and mutants thereof and the CH2 region;

(6) the hinge region of antibody IgG1 and its mutants, the CH2 region and the CH3 region;

(7) hinge region of immunoglobulin Fc receptor: CD64, CD32, CD16, CD89, fceri (CD23), and FcRn;

(8) a CD28 hinge region, a CD137 hinge region, a CD 8a hinge region, a CD4 hinge region, a PD-1 hinge region, and a CTLA-4 hinge region; and

(9) any combination of the above.

The hrCD16 chimeric receptor according to the invention, wherein the extracellular spacer is a CD8 hinge region; preferably, the extracellular spacer region comprises the amino acid sequence shown as SEQ ID NO 5.

The hrCD16 chimeric receptor according to the present invention, wherein the transmembrane region includes, but is not limited to, any one or more of the following: a transmembrane region of the CD3 xi chain of the T cell receptor complex, a CD28 transmembrane region, a CD137 transmembrane region, a CD 8a transmembrane region, a CD4 transmembrane region, a PD-1 transmembrane region, a CTLA-4 transmembrane region, an immunoglobulin Fc receptor transmembrane region, and combinations thereof.

Preferably, the transmembrane region is the CD8 transmembrane region; more preferably, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO 6.

The hrCD16 chimeric receptor according to the present invention, wherein the costimulatory signaling domain includes, but is not limited to, any one or more of the following signaling domains: CD2, CD27, CD28, CD30, CD40, CD40L, CD137(4-1BB), CD134(OX40), CD278(ICOS), GITR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, Dap10, ICAM-1, LFA-1, Lck, TNFRI, TNFRII, TIM-1, TIM-2, TIM-3, TIM-4 and combinations thereof.

Preferably, the co-stimulatory signaling domain is a CD137 signaling domain; more preferably, the co-stimulatory signaling domain comprises the amino acid sequence shown as SEQ ID NO 7.

The chimeric receptor according to the present invention, wherein the cytokine receptor signaling domain includes, but is not limited to, any one or more of the following signaling domains: IL-2R, IL-7R, IL-15R, IL-18R, IL-21R, IL-23R and combinations thereof.

The chimeric receptor according to the present invention, wherein the intracellular signaling domain includes, but is not limited to, any one or more of the following signaling domains: the CD3 ξ chain of the T cell receptor complex, Fc γ RIII, Fc ∈ RI, Fc receptor intracellular signaling domains, signaling domains carrying the Immunoreceptor Tyrosine Activation Motif (ITAM), and combinations thereof.

Preferably, the intracellular signaling domain is a CD3 ξ -chain signaling domain; more preferably, the intracellular signaling domain is represented by SEQ ID NO 8.

Preferably, the receptor consists of a wild type CD16 ectodomain comprising an amino acid sequence as set forth in any of SEQ ID NOs 1-4, a F176V mutant CD16 ectodomain, a S197P mutant CD16 ectodomain, and any of the F176V and S197P double mutant CD16 ectodomains, a human CD8 hinge region, a human CD8 transmembrane region, a human CD137 costimulatory signaling domain, and a CD3 zeta chain signaling domain;

preferably, the amino acid sequence of the chimeric receptor is shown in any one of SEQ ID NO 9-12.

More preferably, the chimeric receptor according to the invention is characterized in that it consists of the F176V and S197P double mutant CD16 ectodomains comprising the amino acid sequence shown in SEQ ID No. 4, a human CD8 hinge region, a human CD8 transmembrane region, a human CD137 costimulatory signaling domain and a CD3 ξ chain signaling domain.

More preferably, the amino acid sequence of the double mutant hrCD16 chimeric receptor is shown as SEQ ID NO. 12.

The invention also provides a polynucleotide encoding the chimeric receptor.

Preferably, the polynucleotide sequence is as shown in any one of SEQ ID NO 13-16;

more preferably, the polynucleotide sequence is as shown in SEQ ID NO 16

The invention also provides a vector comprising the polynucleotide.

Preferably, the vector co-expresses a cytokine, chemokine receptor, immune checkpoint blocking antibody, or a combination thereof;

more preferably, the cytokines include, but are not limited to, IL-2, IL-7, IL-15, IL-21, IL-12, IL-18, IL-23 and combinations thereof; such chemokines include, but are not limited to CXCL9, CXCL10, CXCL11, CCL19, CCL20, and CCL 21; the chemokine receptors include, but are not limited to, CCR1, CCR3, CCR9, CXCR1, and CXCR 2; the immune checkpoint blocking antibodies include, but are not limited to, CTLA-4 blocking antibodies, PD-1 blocking antibodies, PD-L1 blocking antibodies, LAG-3 blocking antibodies, Tim-3 blocking antibodies, TIGIT blocking antibodies, VISTA blocking antibodies, Siglec-15 blocking antibodies, and combinations thereof.

The invention also provides a virus comprising the polynucleotide;

preferably, the virus includes, but is not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus, poxvirus, and herpesvirus.

In another aspect, the invention provides an immune cell expressing the chimeric receptor.

Preferably, the immune cells include, but are not limited to, T cells, Natural killer cells (NK), intrinsic lymphocytes (incnate lymphoid cells, ILC), hematopoietic stem cells, embryonic stem cells, pluripotent stem cells, and the like;

more preferably, the T cells include, but are not limited to, unsorted and purified T cells, sorted and purified PD-1⁺T cell, sorting purified CD137⁺T cell, sorting purified CD160⁺T cell, sorting purified pure T cell (T)_naive) Sorting purified central memory T cells (T)_CM) Sorting purified effector memory T cells (T)_EM) Sorting purified effector T cells (T)_EMRA) Sorting purified T cells of Transitional Memory type (T cells, T)_TM) Sorting purified Tissue memory T cells (T cells, T)_RM) And Natural killer T cells (NKT), and the like.

In yet another aspect, the invention provides a combination comprising said immune cell and a tumor antigen targeting antibody.

Preferably, the tumor antigens include, but are not limited to, one or more of the following: CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD70, CD117, CD123, CD133, CD138, CD147, CD171, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13R α 2, EGFR, EGFRvIII, GD2, DR5, EphA2, FR α, PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, MeOthelin, DLL3 and CLDN 18.

More preferably, the tumor antigens include, but are not limited to, one or more of the following: HER2, EGFR, CD47, AXL and FAP.

In yet another aspect, the invention provides a combination comprising said immune cell and a viral antigen targeting antibody.

Preferably, the viral antigen is selected from one or more of the following: gp120 of human acquired immunodeficiency virus HIV-1, surface antigen of hepatitis B virus HBV, hemagglutinin or neuraminidase of influenza virus, spike protein of Ebola virus, surface spike protein of severe acute respiratory syndrome coronavirus SARS-CoV, surface spike protein of middle east respiratory syndrome coronavirus MERS-CoV and surface spike protein of novel coronavirus SARS-CoV-2;

more preferably, the viral antigen is selected from the surface spike protein of the novel coronavirus SARS-CoV-2.

In still another aspect, the present invention provides the use of the chimeric receptor, the immune cell, the combination of the immune cell and the tumor antigen-targeting antibody, and the combination of the immune cell and the virus antigen-targeting antibody of the present invention in the preparation of a medicament for treating a tumor or a virus-infectious disease;

preferably, the tumour is selected from one or more of: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, rhabdomyoma and leiomyoma;

preferably, the viral infectious disease is selected from one or more of the following: human acquired immunodeficiency syndrome, hepatitis b, influenza, ebola virus disease, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and novel coronavirus pneumonia.

In yet another aspect, the invention provides a method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of a combination of a chimeric receptor, an immune cell, and a tumor antigen-targeting antibody;

preferably, the tumour is selected from one or more of: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, rhabdomyoma, and leiomyoma.

In a preferred embodiment, the invention provides a method of treating FAP + tumors comprising administering to a patient a combination of an immune cell of the invention and a FAP-targeting antibody.

In a preferred embodiment, the invention provides a method of treating AXL + tumors comprising administering to a patient a combination of an immune cell of the invention and an AXL-targeting antibody.

In a preferred embodiment, the present invention provides a method of treating a CD47+ tumor, the method comprising administering to a patient a combination of an immune cell of the invention and a CD47 targeting antibody. In a preferred embodiment, the invention provides a method of treating HER2+ tumor comprising administering to a patient a combination of an immune cell of the invention and a HER2 targeting antibody.

In a preferred embodiment, the invention provides a method of treating an EGFR + tumor comprising administering to a patient a combination of an immune cell of the invention and an EGFR-targeting antibody.

In yet another aspect, the invention provides a method of treating a viral infectious disease, the method comprising administering to a subject a therapeutically effective amount of a combination of immune cells, immune cells and a viral antigen-targeting antibody;

preferably, the virus is selected from one or more of the following: human acquired immunodeficiency virus HIV-1, hepatitis B virus HBV, influenza virus, Ebola virus, severe acute respiratory syndrome coronavirus SARS-CoV, middle east respiratory syndrome coronavirus MERS-CoV and novel coronavirus SARS-CoV-2.

In a preferred embodiment, the present invention provides a method of treating a novel coronavirus SARS-CoV-2 infectious disease, said method comprising administering to a patient a combination of an immune cell of the invention and a SARS-CoV-2 surface spike protein targeting antibody.

In still another aspect, the present invention provides a method for preparing the immune cell of the present invention, which comprises the steps of:

1) obtaining a nucleic acid sequence of the chimeric receptor;

2) cloning the nucleic acid sequence of the chimeric receptor into a lentivirus expression vector to obtain a lentivirus expression plasmid for coding the chimeric receptor;

3) cotransfecting the lentivirus expression plasmid, the skeleton plasmid and the envelope plasmid to HEK293T cells, packaging and obtaining lentivirus particles, and obtaining a lentivirus concentrated solution after centrifugal concentration;

4) transducing a lentivirus into an immune cell, thereby obtaining an immune cell expressing a chimeric receptor;

preferably, the immune cell is a T cell.

The present invention also provides a method for expanding a plurality of T cells expressing a chimeric receptor, the method comprising: transfecting T cells with said vector or infecting T cells with a virus as described; and the additional addition of anti-human CD3 stimulating antibodies and anti-human CD28 stimulating antibodies, tumor antigen expressing cells or recombinant tumor antigens, and tumor antigen targeting antibodies to stimulate the T cells to proliferate in large numbers to produce large numbers of chimeric receptor engineered T cells.

Compared with the prior art, the invention has the following advantages:

1) the invention provides a novel Fc high-affinity cell protease-tolerant chimeric receptor, which has higher affinity of an antibody Fc fragment, is used in combination with a tumor antigen targeting antibody, and can remarkably enhance ADCC (ADCC) effect.

2) The chimeric receptor of the invention can resist the cutting of metalloprotease, ensure the high-efficiency expression of the chimeric receptor on killer cells and avoid the function damage caused by the down regulation of the chimeric receptor.

3) The chimeric receptor of the invention is different from the traditional single-target CAR strategy, and can flexibly switch the target antibodies of different tumor antigens, thereby realizing the efficacy of broad-spectrum immunotherapy of multiple cancer species.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a recombinant lentivirus expression plasmid map

FIG. 2 is a graph showing the effect of the chimeric receptor-binding antibody Fc fragment according to the present invention.

FIG. 3 is a graph showing the expression of chimeric receptors according to the invention on the surface of primary T cell membranes.

Fig. 4 is a graph of tumor cell killing by chimeric receptor-expressing T cells in combination with FAP-targeting antibodies according to the present invention.

Figure 5 is a graph of killing of tumor cells by chimeric receptor expressing T cells in combination with AXL targeting antibodies according to the present invention.

Fig. 6 is a graph of the killing of tumor cells by chimeric receptor-expressing T cells in combination with CD47 targeting antibodies according to the present invention.

FIG. 7 is a graph of the efficiency of killing tumor cells by T cells expressing a wild-type/single point mutation/double mutation chimeric receptor in combination with a CD47 targeting antibody and a statistical plot of luciferase expression levels of surviving tumor cells according to the present invention;

figure 8 is a graph of the efficiency of chimeric receptor expressing T cells in combination with HER2 targeting antibodies in killing tumor cells according to the invention.

Fig. 9 is a graph of the efficiency of killing tumor cells by T cells expressing wild type/single point mutation/double mutation chimeric receptors in combination with HER2 targeting antibody and a statistical graph of luciferase expression levels of surviving tumor cells.

FIG. 10 is a graph of the efficiency of T cells expressing chimeric receptors in killing tumor cells in combination with EGFR-targeting antibodies according to the present invention.

FIG. 11 is a graph showing the efficiency of T cells expressing chimeric receptors in combination with SARS-CoV-2 surface Spike protein targeted antibodies in killing Spike protein positive A549-Spike cells according to the present invention.

FIG. 12 is a graph of tumor growth inhibition by chimeric receptor-expressing T cells in combination with CD 47-targeted antibodies according to the present invention.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

The experimental procedures of the following examples are, unless otherwise specified, routine in the art. The experimental materials used in the following examples were, unless otherwise specified, purchased from conventional biochemical reagents sales companies, among which:

DMEM medium and RPMI1640 medium were purchased from Corning, and lymphocyte culture medium X-VIVO 15 was purchased from Lonza.

The T cell growth medium consists of a basic medium and cytokines, wherein the basic medium is lymphocyte culture medium X-VIVO 15, and the cytokines are IL-7 with the final concentration of 5ng/mL, IL-15 with the final concentration of 10ng/mL and IL-21 with the final concentration of 30 ng/mL. Among them, cytokines IL-7 and IL-15 were purchased from R & D, and IL-21 was purchased from near-shore protein science and technology Co.

Fetal bovine serum was purchased from BI corporation.

TurboFect transfection kit was purchased from Thermo Fisher Scientific.

Lenti-X lentivirus concentration reagents were purchased from Takara.

Tumor-targeting antibodies and novel coronavirus SARS-CoV-2 surface spike protein-targeting antibodies were prepared and provided by Shanghai Xinwan Biotech Co., Ltd., and include HER 2-targeting antibody (XW-HER2-02), EGFR-targeting antibody (XW-EGFR-02), CD47(XW-CD47-02), GPC 3-targeting antibody (XW-GPCR-02), AXL-targeting antibody (XW-AXL-02), FAP-targeting antibody (XW-FAP-02) and SARS-CoV-2 surface spike protein-targeting antibody (XW-SARS-CoV-2).

Synthetic genes were purchased from Shanghai Czeri bioengineering, Inc.

Lentiviral expression plasmid pXW-EF1 α -MCS-P2A-EGFP was supplied by Shanghai Xinwan Biotech, Inc., and packaging plasmid psPAX2 and envelope plasmid PMD2.G were purchased from Addgene.

Stable 3 chemically competent cells were purchased from shanghai medico biotechnology limited.

The endotoxin-free plasmid miniprep kit and the endotoxin-free plasmid midprep kit were purchased from OMEGA and Macherey Nagel, respectively.

Luciferase substrates were purchased from Promega Biotechnology Ltd.

HEK293T cells, A549 lung cancer cells, NCI-H292 high-metastasis lung cancer cells, U251 glioma cells, MDA-MB-231 breast cancer cells, HepG2 liver cancer cells were purchased from American ATCC. A549-luc lung cancer cell, NCI-H292-luc high-metastasis lung cancer cell, U25-luc glioma cell, MDA-MB-231-luc breast cancer cell, HepG2-luc liver cancer cell and A549-FAP cell are purchased from Shanghai Xinwan Biotech limited.

Real-time label-free cell function analyzers (RTCAs) were purchased from shanghai goodpaste biotechnology limited.

The microplate luminescence detector was purchased from Promega Biotechnology Ltd.

B-NDG immunodeficient mice were purchased from Beijing Baiosaoxi map Gene Biotechnology, Inc.

Unless otherwise specified, in the following examples, the hrCD16 chimeric receptor represents the F176V and S197P double mutant CD16 chimeric receptor according to the invention; the CD16 chimeric receptor represents a chimeric receptor containing the wild-type CD16, the CD16F176V chimeric receptor represents a chimeric receptor containing the F176V mutated CD16, and the CD16S197P chimeric receptor represents a chimeric receptor containing the S197P mutated CD16.EXAMPLE 1 construction of lentivirus expression plasmids

Wild-type CD16.BBz, F176V mutant CD16.BBz, S197P mutant CD16.BBz and hrCD16.BBz genes (shown as SEQ ID NO:13-16, respectively) were synthesized by Shanghai Czeri bioengineering, Inc., and cloned into blank lentiviral expression plasmids (pXW-EF1 α -MCS-P2A-EGFP) to obtain pXW-EF1 α -CD16.BBz-P2A-EGFP, pXW-EF1 α -CD1F176V.BBz-P2A-EGFP, pXW-EF1 α -CD169650 S197P.BBz-P2A-EGFP and pXW-EF1 α -CD16.BBz-P2A-EGFP recombinant lentiviral expression plasmids, and the plasmid maps are shown in FIG. 1.

Example 2 lentivirus expression plasmid transfection of HEK293T cells

One day before the experiment, HEK293T cells, 4X 10 cells, were seeded on 12-well flat-bottomed cell culture plates⁵Cells/2 mL/well. The following day, the lentiviral expression plasmids pXW-CD16.BBz and XW-hrCD16.BBz were transfected separately by TurboFect transfection reagent, totaling 2. mu.g/well. In plasmid amount (μ g): transfection reagent (μ L) ═ 1: 2, adding 4 mu L of TurboFect transfection reagent, incubating the freshly prepared plasmid transfection complex at room temperature for 15-20 min, and adding the incubated plasmid transfection complex into the cell culture plate. Standing at 37 deg.C for 5% CO₂Culturing for 48 hr at 500 Xg, centrifuging at room temperature for 5min, removing supernatant, and collecting cells.

Example 3 efficient binding of the chimeric receptor hrCD16 to the Fc fragment of an antibody

HEK293T cells expressing the wild-type CD16 chimeric receptor (cd16.bbz) and the hrCD16 chimeric receptor (hrcd16.bbz) were prepared separately using the method described in example 2.

Respectively taking 1 × 10⁵Placing wild type CD16 chimeric receptor and hrCD16 chimeric receptor modified HEK293T cells into a 1.5mL EP tube, adding EGFR targeting antibody labeled by fluorescent dye AF647, adjusting the working concentration of the antibody to be 0.5, 1 and 5 mu g/mL, incubating for 30min at 4 ℃ in a dark place, eluting for 2 times by FACS buffer (1 XPBS containing 2% FBS), and detecting by using a flow cytometer after resuspending by 200-300 mu LFACS buffer.

The results are as follows: fig. 2A is a flow chart of binding of HEK293T cells expressing the wild-type CD16 chimeric receptor and the hrCD16 chimeric receptor to the Fc fragment of the EGFR-targeting antibody, respectively. At an action concentration of 5. mu.g/mL of antibody, HEK293T cells expressing the hrCD16 chimeric receptor bound the Fc fragment of the EGFR targeting antibody far more efficiently than HEK293T cells expressing the wild type CD16 chimeric receptor (percentage: 85.8% vs 40.4%; fluorescence intensity: 3720vs 1033). Fig. 2B is a statistical plot of the efficiency of binding of HEK293T cells expressing the wild-type CD16 chimeric receptor and HEK293T cells expressing the hrCD16 chimeric antibody to the Fc fragment of the EGFR-targeting antibody. On the binding rate and strength of the Fc fragment, the ability of HEK293T cells expressing the hrCD16 chimeric receptor to bind the antibody Fc fragment is superior to that of HEK293T cells expressing a wild-type CD16 chimeric antibody, and particularly, the difference is obvious under the condition of higher antibody action concentration.

Example 4 packaging, concentration and titer determination of lentiviruses

1.1 packaging of lentiviruses

HEK293T cell treatment: 24 hours before transfection, HEK293T cells in logarithmic growth phase were collected and seeded in 10cm cell culture dishes (6-8X 10)⁶Individual cells), cells were grown in complete DMEM medium containing 10mL, placed at 37 ℃, 5% CO₂And culturing for 18-24 hours under the condition, and performing transfection when the cell density reaches more than 70-90%.

HEK293T cell transfection: adding 1mL of basic DMEM medium into a 15mL centrifuge tube, and preparing a transfection mixed solution according to the mass ratio of the slow virus expression plasmid psPAX2 to the envelope plasmid PMD2G to 1:3:1, wherein the total amount of the plasmids is 15 mu g/dish. In plasmid amount (μ g): transfection reagent (μ L) ═ 1: 2, adding 30 mu L of TurboFect transfection reagent, incubating at room temperature for 15-20 min, adding into a cell culture dish, placing at 37 ℃ and 5% CO₂Culturing in a cell culture box for 48 hours, collecting virus supernatant, centrifuging at 1000 Xg and 4 ℃ for 10min, and collecting supernatant virus.

1.2 concentration of lentiviruses

And filtering the centrifugally collected virus supernatant by using a 0.45-micron filter, adding a Lenti-X lentivirus concentration reagent with the volume of 1/3 virus supernatant, reversing and uniformly mixing for several times, incubating overnight at 4 ℃, centrifuging for 45min at 2000 Xg and 4 ℃, and obtaining the virus after white precipitate is visible at the bottom of a centrifugal tube. Carefully discard the supernatant, resuspend the white pellet in 1/50-1/100 volumes of blank RPMI1640 medium from the proviral supernatant, split and freeze at-80 ℃ for use.

1.3 lentivirus titer assay Jurkat T cells were assayed at 1X 10⁵One/well was seeded on a 96-well U-plate and the collected lentivirus concentrates were diluted in 10-fold increments. Adding 100 mu L of virus diluent into a corresponding hole, adding protamine sulfate serving as an infection promoting reagent, regulating the concentration to 10 mu g/mL, 1000 Xg, carrying out centrifugal infection at 32 ℃ for 90min, after overnight culture, replacing the culture solution, continuing to culture for 48 hours, detecting the proportion of fluorescence positive cells by a flow cytometer, and calculating the virus titer by adopting the following formula: viral titer (TU/mL) 1 × 10⁵X fluorescence positive cell ratio/100 x 1000 x corresponding dilution factor.

Example 5 infection and expansion of T cells

In 48-well flat-bottomed cell culture plates (containing 1X 10)⁶Peripheral blood mononuclear cells previously activated), the concentrated lentiviral vector packaged in example 4 (LV-CD16BBz, LV-CD16F176V, LV-CD16S197P or LV-hrCD16.BBz) (MOI 5-10) was added, 10. mu.g/mL of protamine sulfate as an infection-promoting agent was added, 1000 Xg was added, and the mixture was centrifuged at 32 ℃ for 90min and cultured overnight. The next day, the culture medium was changed to fresh T cell growth medium and the culture continued. Adding fresh T cell growth medium every 2-3 days, and adjusting the cell density to 0.5-2 × 10⁶And (4) cells. And (3) removing the immunomagnetic beads of the activated T cells 6-7 days after infection, continuously culturing and amplifying the T cells modified by the hrCD16 chimeric receptor, and performing subsequent functional experiments after the cells are rested (the immunomagnetic beads are removed for 6-7 days).

Example 6 efficient expression of the chimeric receptor hrCD16 on the surface of primary T cells

Primary T cells modified to express hrCD16 chimeric receptor (hrcd16.bbz) were prepared using the method described in example 4.

Take 1X 10⁵Each untransduced T cell/hrCD 16-modified T cell was placed in a 1.5mL EP tube, 0.5. mu.L of flow antibody PerCP/Cy5.5-anti-human CD16 was added, and the tube was incubated at room temperature in the dark for 20min, after which time FACS buffer (2% FBS-containing solution)1 × PBS) for 2 times, and detecting by using a flow cytometer after 200-300 μ L of FACS buffer is resuspended.

The results show that: fig. 3A is a flow chart of expression of hrCD16 on primary T cells. Three human-derived T cells modified by the hrCD16 chimeric receptor normally express hrCD16, the positive rate is 33.9-50.6%, and the fluorescence intensity is 3021-4987 (FIG. 3B).

Example 7T cells expressing hrCD16 chimeric receptor in combination with high-killing FAP-targeting antibody kill FAP + tumor cells Cell

The tumor cell killing efficiency was measured by Real-Time label-free cell Analysis (RTCA) technique. First, 1 × 10⁴A549-FAP (FAP-modified human lung cancer cells) was seeded in a 16-well E-Plate electrode Plate at 100. mu.L per well. And dynamically monitoring the cell growth for 8-9 hours by using an RTCA system. The ratio of effector cells: target cells 3:1, adding T cells expressing the hrCD16 chimeric receptor into a hole containing target cells, respectively adding FAP targeting antibody and high-killing FAP targeting antibody, adjusting the working concentration of the antibody to be 1 mu g/mL, recording the measurement result every 15 minutes, and continuously recording for 24 hours.

The results are shown in FIG. 4: fig. 4 shows that the solid line is the target cell growth curve of the tumor cell killed by the T cell expressing the hrCD16 chimeric receptor and the FAP-targeting antibody, and the dotted line is the target cell growth curve of the tumor cell killed by the T cell expressing the hrCD16 chimeric receptor, and the result shows that the difference between the target cell growth curve of the T cell expressing the hrCD16 chimeric receptor and the FAP-targeting antibody set and the target cell growth curve of the T cell expressing the hrCD16 chimeric receptor is significant, which indicates that the tumor cell can be killed efficiently by the T cell expressing the hrCD16 chimeric receptor and the FAP-targeting antibody, the tumor cell killing rate is as high as 100%, and the growth of the tumor cell is effectively inhibited, so that the tumor cell growth curve is rapidly reduced.

Example 8T cells expressing the hrCD16 chimeric receptor in combination with highly lethal AXL-targeting antibodies kill AXL + tumor cells Cell

Tumor cell killing efficiency by real-time label-free cell analysis (RTC)A, Real Time Cellular Analysis) technique. First, 1 × 10⁴Each U251 (human glioma cells) or MDA-MB-231 (human breast cancer cells) was seeded on a 16-well E-Plate electrode Plate with 100. mu.L of medium per well. Cell growth was monitored dynamically using an RTCA system for 18-20 hours. The ratio of effector cells: target cells 3:1 into wells containing target cells and highly lethal AXL targeting antibody, the working concentration of antibody was adjusted to 1 μ g/mL, the assay results were recorded every 15 minutes for 24 hours.

The results are shown in FIG. 5: fig. 5A shows that the solid line is the growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the AXL targeting antibody to kill the U251 tumor cell, the dotted line is the growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the irrelevant antibody to kill the U251 tumor cell, and the other two curves are the growth curve of the U251 tumor cell and the growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the AXL targeting antibody, respectively, and the results show that the U251 tumor cell growth curve can be rapidly decreased only by the T cell expressing the hrCD16 chimeric receptor in combination with the AXL targeting antibody, and the killing rate is as high as 94.5%; FIG. 5B shows the solid line of the MDA-MB-231 tumor cell growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the AXL targeting antibody, the dotted line of the MDA-MB-231 tumor cell growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the irrelevant antibody, and the other two curves of the MDA-MB-231 tumor cell growth curve and the MDA-MB-231 tumor cell growth curve of the T cell expressing the hrCD16 chimeric receptor, respectively, and only the T cell expressing the hrCD16 chimeric receptor in combination with the MDA-MB-231 targeting antibody can rapidly decrease the MDA-MB-231 tumor cell growth curve, effectively kill the tumor cells, and the killing rate is as high as 91.8%.

Example 9 killing of CD47+ tumors by T cells expressing hrCD16 chimeric receptor in combination with highly lethal CD47 targeting antibody Cells

The tumor cell killing efficiency was measured by using Real Time label free cell Analysis (RTCA) technique. First, 1 × 10⁴Each U251 (human glioma cells) or MDA-MB-231 (human breast cancer cells) was seeded on a 16-well E-Plate electrode Plate with 100. mu.L of medium per well. Cell growth was monitored dynamically using an RTCA system for 18-20 hours. The ratio of effector cells: target cells 3:1 into wells containing target cells and a highly lethal CD47 targeting antibody, the working concentration of the antibody was adjusted to 1 μ g/mL, and the assay results were recorded every 15 minutes for 24 hours.

As shown in fig. 6, the solid line in fig. 6A is the target cell growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the CD47 targeted antibody to kill the U251 tumor cell, the dotted line is the target cell growth curve of the T cell expressing the hrCD16 chimeric receptor in combination with the unrelated antibody to kill the U251 tumor cell, and the other two curves are the growth curve of the U251 tumor cell and the target cell growth curve of the T cell expressing the hrCD16 chimeric receptor to kill the tumor cell, respectively, which shows that only the T cell expressing the hrCD16 chimeric receptor in combination with the CD47 targeted antibody can rapidly decrease the U251 tumor cell growth curve to effectively kill the tumor cell, with a killing rate as high as 94.2%; FIG. 6B shows the solid line of the growth curve of MDA-MB-231 tumor cells killed by the T cells expressing the hrCD16 chimeric receptor in combination with the CD47 targeting antibody, the dotted line of the growth curve of MDA-MB-231 tumor cells killed by the T cells expressing the hrCD16 chimeric receptor in combination with the irrelevant antibody, and the other two curves of MDA-MB-231 tumor cells and the growth curve of MDA-MB-231 tumor cells killed by the T cells expressing the hrCD16 chimeric receptor, respectively, which shows that only the T cells expressing the hrCD16 chimeric receptor in combination with the MDA-MB-231 targeting antibody can rapidly decrease the MDA-MB-231 tumor cell growth curve and effectively kill the tumor cells.

Example 10T cells expressing different chimeric receptors in combination with a highly lethal CD47 targeting antibody kill CD47+ tumors Cells

The tumor cell killing efficiency was evaluated by using a Luciferase-based cell killing assay (Luciferase-based cytotoxin assay). First, 1 × 10⁴SKOV3-Luc (luciferase gene modified human ovarian carcinoma cells) was inoculated100. mu.L of medium per well in 96-well flat bottom plates at 37 ℃ in 5% CO₂Culturing for 18-20 hours in a cell culture box. The following day, the ratio of effector cells: target cells ═ 1: 1 into wells containing target cells, including CD16.BBz, CD16F176V. BBz, CD1696197P. BBz and hrCD16.BBz, and highly lethal CD47 targeting antibody, adjusted to a working concentration of 2 μ g/mL, placed at 37 ℃ and 5% CO₂Continuously culturing for 18-20 hours in a cell culture box, and using after co-culture is finished

The microplate luminescence detector detects the luciferase activity value of the target cells. The cell killing rate was calculated as follows:

cell killing rate (%) - (luciferase activity value of target cell group-luciferase activity value of experimental group)/luciferase activity value of target cell group × 100

As shown in fig. 7, the CD47 targeting antibody combined with T cells expressing different chimeric receptors can kill CD47+ tumor cells, and the hrCD16 modified T cell treatment group had the least tumor survival (fig. 7A) and the highest tumor cell killing efficiency (fig. 7B), which is superior to wild-type CD16, single mutant CD16F176V and CD16S197P.

Example 11T cells expressing the hrCD16 chimeric receptor in combination with a high-killing HER2 targeting antibody kill HER2+ tumors Tumor cell

The tumor cell killing efficiency was evaluated by using a Luciferase-based cell killing assay (Luciferase-based cytotoxin assay). First, 1 × 10⁴NCI-H292-Luc (luciferase gene modified human lung cancer cells) or SKOV3-Luc (luciferase gene modified human ovarian cancer cells) were inoculated on a 96-well flat-bottom plate at a temperature of 37 ℃ and 5% CO in 100. mu.L per well₂Culturing for 18-20 hours in a cell culture box. The following day, the ratio of effector cells: target cells 3:1, adding T cells expressing hrCD16 chimeric receptor into the wells containing target cells, adding high-killing HER2 targeting antibody with working concentration of 1 μ g/mL, placing at 37 deg.C and 5% CO₂Continuously culturing for 18-20 hours in a cell culture box, and using after co-culture is finished

As shown in fig. 8, the T cells expressing the hrCD16 chimeric receptor had some non-specific killing effect on tumor cells, while the killing efficiency of the group with the irrelevant control antibody was comparable to that of the group with the hrCD16 chimeric receptor alone, indicating that ADCC effect of the irrelevant control antibody was extremely weak. The HER2 targeting antibody and the T cell expressing the hrCD16 chimeric receptor can effectively kill tumor cells, and the killing rates of lung cancer cells (NCI-H292) and ovarian cancer cells (SKOV3) are respectively as high as 92.2% and 89.4%, so that the tumor cells are effectively eliminated.

Example 12T cells expressing different chimeric receptors in combination with a high killing HER2 targeting antibody kill HER2+ tumors Cells

The tumor cell killing efficiency was evaluated by using a Luciferase-based cell killing assay (Luciferase-based cytotoxin assay). First, 1 × 10⁴SKOV3-Luc (luciferase gene modified human ovarian carcinoma cells) was inoculated on a 96-well flat-bottom plate at a temperature of 37 ℃ and 5% CO in 100. mu.L of each well₂Culturing for 18-20 hours in a cell culture box. The following day, the ratio of effector cells: target cells ═ 1: 1 into wells containing target cells, including CD16.BBz, CD16F176V. BBz, CD1696197P. BBz and hrCD16.BBz, and highly lethal CD47 targeting antibody, adjusted to a working concentration of 2 μ g/mL, placed at 37 ℃ and 5% CO₂Continuously culturing for 18-20 hours in a cell culture box, and using after co-culture is finished

As shown in fig. 9, HER 2-targeted antibody combined with T cells expressing different chimeric receptors can kill HER2+ tumor cells, and hrCD 16-modified T cell treated group had the least tumor survival (fig. 8A) and the highest tumor cell killing efficiency (fig. 8B), superior to wild-type CD16, single mutant CD16F176V, and CD16S197P.

Example 13 killing of tumor cells by T cells expressing the hrCD16 chimeric receptor in combination with highly lethal EGFR-targeting antibodies

The tumor cell killing efficiency was evaluated by using a Luciferase-based cell killing assay (Luciferase-based cytotoxin assay). First, 1 × 10⁴NCI-H292-Luc (luciferase gene modified human lung cancer cells) or SKOV3-Luc (luciferase gene modified human ovarian cancer cells, EGFR +) were inoculated on a 96-well flat-bottom plate at 37 ℃ in 100. mu.L of medium per well with 5% CO₂Culturing for 18-20 hours in a cell culture box. The following day, the ratio of effector cells: target cells 3:1 into the wells containing the target cells, and adding highly lethal EGFR-targeting antibody, the working concentration of the antibody is adjusted to 1 μ g/mL, and the wells are placed at 37 ℃ and 5% CO₂Continuously culturing for 18-20 hours in a cell culture box, and using after co-culture is finished

As shown in fig. 10, the T cells expressing the hrCD16 chimeric receptor had some non-specific killing effect on tumor cells, while the killing efficiency of the group with the irrelevant control antibody was comparable to that of the group with the hrCD16 chimeric receptor alone, indicating that ADCC effect of the irrelevant control antibody was extremely weak. The modified high-killing EGFR targeting antibody can effectively kill tumor cells by combining with T cells expressing the hrCD16 chimeric receptor, and the killing rates of lung cancer cells and ovarian cancer cells are respectively as high as 92.2 percent and 86.1 percent, so that the tumor cells are effectively eliminated.

Example 14T cells expressing the chimeric receptor for hrCD16 in combination with SARS-CoV-2 spike protein-targeted antibody killing Spike + cells

The cell killing efficiency was measured by Real Time unlabeled cell Analysis (RTCA) technique. First, 1 × 10⁴Each Spike protein positive A549-Spike cell was inoculated on a 16-well E-Plate electrode Plate with 100. mu.L of medium per well. Cell growth was monitored dynamically using an RTCA system for 18-20 hours. The ratio of effector cells: target cells 2: 1 into wells containing target cells, and SARS-CoV-2 spike protein targeting antibodies SARS-CoV-2-505-5 and SARS-CoV-2-553-20 were added, the working concentration of the antibody was adjusted to 1. mu.g/mL, and the results of the assay were recorded every 15 minutes for 24 hours.

The results are shown in FIG. 11: t cells expressing the chimeric receptor hrCD16 have a certain non-specific killing effect (40%) against the Spike protein positive A549-Spike. SARS-CoV-2 Spike protein target antibody SARS-CoV-2-505-5 and SARS-CoV-2-553-20 combined expression hrCD16 chimeric receptor T cell can effectively kill Spike protein positive A549-Spike cell, and target cell killing rate is respectively up to 84%.

Example 15 in vivo anti-tumor Effect of T cells expressing the chimeric receptor of hrCD16 in combination with highly lethal CD 47-targeting antibody

NCI-H292-Luc Lung cancer cells were inoculated into the lower left flank of B-NDG immunodeficient mice at 2X 10⁶For each cell, after the tumor grows for 4 days, dividing the tumor-bearing mice into 3 groups randomly, and injecting T cells expressing the hrCD16 chimeric receptor into 3 groups; CD 47-targeted antibody in combination with untransduced T cell injection group, 3; CD47 targetingThe antibody was combined with the hrCD16 chimeric receptor expressing T cell injection group, 3. And monitoring the growth condition of the tumor by using a small animal living body imaging system.

The administration method comprises the following steps: antibody administration: each mouse was injected intraperitoneally with 50 μ g (125 μ L) each time, on days 4, 7 and 18 after tumor inoculation, respectively. Cell administration: each mouse was injected intraperitoneally with 2X 10T cells that did not transduce T cells or express the hrCD16 chimeric receptor⁶Each 125 μ L, was reinfused 3 times a day after antibody administration.

The results are shown in fig. 12, and the abdominal tumor growth of the T cell injection group of the CD47 targeting antibody combined with the hrCD16 chimeric receptor is obviously inhibited, and one tumor-bearing mouse is cured. The abdominal tumors in the remaining two groups continued to grow over time (fig. 12A). The CD 47-targeted antibody was totally alive in combination with the hrCD16 chimeric receptor expressing T cell injected group of mice, while the remaining two groups of mice were totally dead on day 40 of tumor inoculation (fig. 12B).

Although only examples of specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are all within the scope of the invention.

Sequence listing

<110> Shanghai Xinwan Biotech Co., Ltd

<120> a chimeric receptor and uses thereof

<130> DIC20110036R

<150> 2020106405261

<151> 2020-07-06

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Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

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Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

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Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

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Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

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Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

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Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

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Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

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Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

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Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

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Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

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Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

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Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

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Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

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His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

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Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

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

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

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Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

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Ser Leu Val Ile Thr Leu Tyr Cys

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

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

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

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

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

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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

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

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Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

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Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

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Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

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Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

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Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

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Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

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Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

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Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

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

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Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

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Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

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Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

245 250 255

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

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Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

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Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

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Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

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

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

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

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

370 375 380

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

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

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

420 425 430

Arg

<210> 10

<211> 433

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

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

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

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

195 200 205

Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

210 215 220

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

225 230 235 240

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

245 250 255

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

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Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

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Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

290 295 300

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Arg

<210> 11

<211> 433

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

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

195 200 205

Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

210 215 220

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

225 230 235 240

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

245 250 255

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

260 265 270

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

275 280 285

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

290 295 300

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Arg

<210> 12

<211> 433

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

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

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

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

195 200 205

Thr Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

210 215 220

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

225 230 235 240

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

245 250 255

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

260 265 270

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

275 280 285

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

290 295 300

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Arg

<210> 13

<211> 1299

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgtggcagc tgctgctgcc caccgccctg ctgctgctgg tgagcgccgg catgcgcacc 60

gaggacctgc ccaaggccgt ggtgttcctg gagccccagt ggtaccgcgt gctggagaag 120

gacagcgtga ccctgaagtg ccagggcgcc tacagccccg aggacaacag cacccagtgg 180

ttccacaacg agagcctgat cagcagccag gccagcagct acttcatcga cgccgccacc 240

gtggacgaca gcggcgagta ccgctgccag accaacctga gcaccctgag cgaccccgtg 300

cagctggagg tgcacatcgg ctggctgctg ctgcaggccc cccgctgggt gttcaaggag 360

gaggacccca tccacctgcg ctgccacagc tggaagaaca ccgccctgca caaggtgacc 420

tacctgcaga acggcaaggg ccgcaagtac ttccaccaca acagcgactt ctacatcccc 480

aaggccaccc tgaaggacag cggcagctac ttctgccgcg gcctgttcgg cagcaagaac 540

gtgagcagcg agaccgtgaa catcaccatc acccagggcc tggccgtgag caccatcagc 600

agcttcttcc cccccggcta ccagacccgc accaccaccc ccgccccccg cccccccacc 660

cccgccccca ccatcgccag ccagcccctg agcctgcgcc ccgaggcctg ccgccccgcc 720

gccggcggcg ccgtgcacac ccgcggcctg gacttcgcct gcgacatcta catctgggcc 780

cccctggccg gcacctgcgg cgtgctgctg ctgagcctgg tgatcaccct gtactgcaag 840

cgcggccgca agaagctgct gtacatcttc aagcagccct tcatgcgccc cgtgcagacc 900

acccaggagg aggacggctg cagctgccgc ttccccgagg aggaggaggg cggctgcgag 960

ctgcgcgtga agttcagccg cagcgccgac gcccccgcct acaagcaggg ccagaaccag 1020

ctgtacaacg agctgaacct gggccgccgc gaggagtacg acgtgctgga caagcgccgc 1080

ggccgcgacc ccgagatggg cggcaagccc cgccgcaaga acccccagga gggcctgtac 1140

aacgagctgc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1200

cgccgccgcg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggac 1260

acctacgacg ccctgcacat gcaggccctg cccccccgc 1299

<210> 14

<211> 1299

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgtggcagc tgctgctgcc caccgccctg ctgctgctgg tgagcgccgg catgcgcacc 60

gaggacctgc ccaaggccgt ggtgttcctg gagccccagt ggtaccgcgt gctggagaag 120

gacagcgtga ccctgaagtg ccagggcgcc tacagccccg aggacaacag cacccagtgg 180

ttccacaacg agagcctgat cagcagccag gccagcagct acttcatcga cgccgccacc 240

gtggacgaca gcggcgagta ccgctgccag accaacctga gcaccctgag cgaccccgtg 300

cagctggagg tgcacatcgg ctggctgctg ctgcaggccc cccgctgggt gttcaaggag 360

gaggacccca tccacctgcg ctgccacagc tggaagaaca ccgccctgca caaggtgacc 420

tacctgcaga acggcaaggg ccgcaagtac ttccaccaca acagcgactt ctacatcccc 480

aaggccaccc tgaaggacag cggcagctac ttctgccgcg gcctggtggg cagcaagaac 540

gtgagcagcg agaccgtgaa catcaccatc acccagggcc tggccgtgag caccatcagc 600

agcttcttcc cccccggcta ccagacccgc accaccaccc ccgccccccg cccccccacc 660

cccgccccca ccatcgccag ccagcccctg agcctgcgcc ccgaggcctg ccgccccgcc 720

gccggcggcg ccgtgcacac ccgcggcctg gacttcgcct gcgacatcta catctgggcc 780

cccctggccg gcacctgcgg cgtgctgctg ctgagcctgg tgatcaccct gtactgcaag 840

cgcggccgca agaagctgct gtacatcttc aagcagccct tcatgcgccc cgtgcagacc 900

acccaggagg aggacggctg cagctgccgc ttccccgagg aggaggaggg cggctgcgag 960

ctgcgcgtga agttcagccg cagcgccgac gcccccgcct acaagcaggg ccagaaccag 1020

ctgtacaacg agctgaacct gggccgccgc gaggagtacg acgtgctgga caagcgccgc 1080

ggccgcgacc ccgagatggg cggcaagccc cgccgcaaga acccccagga gggcctgtac 1140

aacgagctgc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1200

cgccgccgcg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggac 1260

acctacgacg ccctgcacat gcaggccctg cccccccgc 1299

<210> 15

<211> 1299

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgtggcagc tgctgctgcc caccgccctg ctgctgctgg tgagcgccgg catgcgcacc 60

gaggacctgc ccaaggccgt ggtgttcctg gagccccagt ggtaccgcgt gctggagaag 120

gacagcgtga ccctgaagtg ccagggcgcc tacagccccg aggacaacag cacccagtgg 180

ttccacaacg agagcctgat cagcagccag gccagcagct acttcatcga cgccgccacc 240

gtggacgaca gcggcgagta ccgctgccag accaacctga gcaccctgag cgaccccgtg 300

cagctggagg tgcacatcgg ctggctgctg ctgcaggccc cccgctgggt gttcaaggag 360

gaggacccca tccacctgcg ctgccacagc tggaagaaca ccgccctgca caaggtgacc 420

tacctgcaga acggcaaggg ccgcaagtac ttccaccaca acagcgactt ctacatcccc 480

aaggccaccc tgaaggacag cggcagctac ttctgccgcg gcctgttcgg cagcaagaac 540

gtgagcagcg agaccgtgaa catcaccatc acccagggcc tggccgtgcc caccatcagc 600

agcttcttcc cccccggcta ccagacccgc accaccaccc ccgccccccg cccccccacc 660

cccgccccca ccatcgccag ccagcccctg agcctgcgcc ccgaggcctg ccgccccgcc 720

gccggcggcg ccgtgcacac ccgcggcctg gacttcgcct gcgacatcta catctgggcc 780

cccctggccg gcacctgcgg cgtgctgctg ctgagcctgg tgatcaccct gtactgcaag 840

cgcggccgca agaagctgct gtacatcttc aagcagccct tcatgcgccc cgtgcagacc 900

acccaggagg aggacggctg cagctgccgc ttccccgagg aggaggaggg cggctgcgag 960

ctgcgcgtga agttcagccg cagcgccgac gcccccgcct acaagcaggg ccagaaccag 1020

ctgtacaacg agctgaacct gggccgccgc gaggagtacg acgtgctgga caagcgccgc 1080

ggccgcgacc ccgagatggg cggcaagccc cgccgcaaga acccccagga gggcctgtac 1140

aacgagctgc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1200

cgccgccgcg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggac 1260

acctacgacg ccctgcacat gcaggccctg cccccccgc 1299

<210> 16

<211> 1302

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgtggcagc tgctgctccc taccgctctc ctgctcctcg tgagcgccgg catgcgcaca 60

gaggacctcc caaaggctgt ggtgttcctg gagcctcagt ggtaccgggt gctcgagaag 120

gactctgtga cactgaagtg ccagggcgct tacagccccg aggacaactc tacccagtgg 180

ttccacaacg agtctctgat ttcttctcag gccagttctt acttcattga cgccgctaca 240

gtggacgaca gcggcgagta cagatgccag acaaacctga gcacactgtc cgaccccgtg 300

cagctggagg tgcacatcgg atggctgctc ctccaggccc ctagatgggt gttcaaggag 360

gaggacccta ttcacctgag atgccactct tggaagaaca ccgccctgca caaggtgaca 420

tacctgcaga acggaaaggg acgcaagtac ttccaccaca actccgactt ctacattcca 480

aaggctacac tgaaggactc tgggtcttac ttctgccgcg gcctcgtggg atctaagaac 540

gtgtctagcg agaccgtgaa cattaccatc acccagggcc tcgccgtgcc aacaattagc 600

agcttcttcc cacccggata ccagacgcgt acaacaaccc cagcccctag gcctccaaca 660

ccagccccaa caatcgcttc tcagccactg tctctcagac ccgaggcttg ccggcctgcc 720

gctggcgggg ccgtgcacac acggggactc gacttcgctt gcgacattta catttgggcc 780

ccactcgctg gaacatgcgg cgtgctcctg ctgtctctgg tgatcacact gtactgcaag 840

cgcgggcgca agaagctgct ctacatcttc aagcagccat tcatgagacc cgtgcagacc 900

acacaggagg aggacggatg ctcttgccgg ttccctgagg aggaggaggg cggatgcgag 960

ctcagagtga agttctctag atctgctgac gccccagctt acaagcaggg gcagaaccag 1020

ctgtacaacg agctcaacct cggccggcgc gaggagtacg acgtgctcga caagcggcgc 1080

ggaagagacc cagagatggg cggaaagcct agaagaaaga accctcagga gggactgtac 1140

aacgagctcc agaaggacaa gatggctgag gcttactccg agattggaat gaagggagag 1200

cggcgcagag gcaaggggca cgacggcctg taccagggac tgtctaccgc caccaaggac 1260

acatacgacg ccctgcacat gcaggccctc ccacctagat ga 1302

Claims

1. A chimeric receptor, characterized in that the chimeric receptor comprises:

(1) the extracellular recognition domain of the Fc fragment;

(2) extracellular spacer;

(3) the transmembrane region; and

(4) intracellular signaling domain;

Optionally, the chimeric receptor further comprises:

(5) one or more costimulatory signaling domains; and/or

(6) one or more cytokine receptor signaling domains;

Wherein, the extracellular recognition domain of the Fc fragment is the CD16 extracellular domain, CD32 extracellular domain, CD64 extracellular domain, CD89 extracellular domain, CD23 extracellular domain, FcεRI extracellular domain, FcRn extracellular domain, Fc binding antibody that specifically binds to the Fc fragment of the antibody , Protein A, Protein G or a mutant thereof or multiple repeating tandem extracellular domains;

Preferably, the extracellular recognition domain of the Fc fragment is the wild-type CD16 extracellular domain, the F176V mutant CD16 extracellular domain, the S197P mutant CD16 extracellular domain or the F176V and S197P double mutant CD16 extracellular domain, which comprises as SEQ ID NO: The amino acid sequence shown in any one of 1-4;

More preferably, the extracellular recognition domain of the Fc fragment is the F176V and S197P double mutant CD16 extracellular domain, which comprises the amino acid sequence shown in SEQ ID NO:4.

2. The chimeric receptor of claim 1, wherein the extracellular spacer comprises one or more selected from the group consisting of:

(1) the hinge region of antibody IgG4 and its mutants;

(2) the hinge region of antibody IgG4 and its mutant and CH2 region;

(3) hinge region of antibody IgG4 and its mutants, CH2 region and CH3 region;

(4) the hinge region of antibody IgG1 and its mutants;

(5) hinge region of antibody IgG1 and its mutant and CH2 region;

(6) hinge region of antibody IgG1 and its mutants, CH2 region and CH3 region;

(7) Hinge region of immunoglobulin Fc receptors: CD64, CD32, CD16, CD89, FcεRI, FcεRII (CD23) and FcRn;

(8) CD28 hinge region, CD137 hinge region, CD8 hinge region, CD4 hinge region, PD-1 hinge region and CTLA-4 hinge region; and

(9) Any combination of the above;

Preferably, the extracellular spacer is a CD8 hinge region; more preferably, the extracellular spacer comprises the amino acid sequence shown in SEQ ID NO: 5;

Preferably, the transmembrane region is selected from one or more of the following: the transmembrane region of the CD3ξ chain of the T cell receptor complex, the CD28 transmembrane region, the CD137 transmembrane region, the CD8 transmembrane region, the CD4 transmembrane region , PD-1 transmembrane region, CTLA-4 transmembrane region, immunoglobulin Fc receptor transmembrane region and combinations thereof; more preferably, the transmembrane region is a CD8 transmembrane region; further preferably, the transmembrane region The membrane region comprises the amino acid sequence shown in SEQ ID NO:6;

Preferably, the costimulatory signaling domain is selected from one or more of the following signaling domains: CD2, CD27, CD28, CD30, CD40, CD40L, CD137, CD134, CD278, GITR, TLR1, TLR2, TLR3 , TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, Dap10, ICAM-1, LFA-1, Lck, TNFRI, TNFRII, TIM-1, TIM-2, TIM-3, TIM-4 and their combination; more preferably, the costimulatory signaling domain is a CD137 signaling domain; further preferably, the costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 7;

Preferably, the cytokine receptor signaling domain is selected from one or more of the following signaling domains: IL-2R, IL-7R, IL-15R, IL-18R, IL-21R, IL-23R and combinations thereof;

Preferably, the intracellular signaling domain is selected from one or more of the following signaling domains: CD3ξ chain of T cell receptor complexes, FcγRIII, FcεRI, Fc receptor intracellular signaling domains, immune-carrying Signal transduction domains of receptor tyrosine activation motif (ITAM) and combinations thereof; more preferably, the intracellular signal transduction domain is a CD3ξ chain signal transduction domain; further preferably, the intracellular signal transduction domain The guide domain is shown in SEQ ID NO:8;

Preferably, the receptor consists of a CD16 extracellular domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-4, a human CD8 hinge region, a human CD8 transmembrane region, a human CD137 costimulatory signaling domain and CD3 ξ chain signaling domain;

Preferably, the amino acid sequence of the chimeric receptor is shown in any one of SEQ ID NOs: 9-12.

More preferably, the receptor is signaled by the CD16 extracellular domain comprising the amino acid sequence shown in SEQ ID NO:4, the human CD8 hinge region, the human CD8 transmembrane region, the human CD137 costimulatory signaling domain, and the CD3 ξ chain Domain composition; more preferably, the amino acid sequence of the chimeric receptor is shown in SEQ ID NO:12.

3. A polynucleotide, wherein the polynucleotide encodes the chimeric receptor of claim 1 or 2;

Preferably, the polynucleotide comprises the nucleotide sequence shown in any one of SEQ ID NOs: 13-16;

More preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 16

4. A carrier comprising the polynucleotide of claim 3;

Preferably, the vector co-expresses cytokines, chemokines, chemokine receptors, immune checkpoint blocking antibodies or a combination thereof;

More preferably, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, IL-12, IL-18, IL-23 and combinations thereof; the chemokine is selected from CXCL9, CXCL10, CXCL11, CCL19, CCL20 and CCL21; the chemokine receptor is selected from CCR1, CCR3, CCR9, CXCR1 and CXCR2; the immune checkpoint blocking antibody is selected from CTLA-4 blocking antibody, PD-1 blocking antibody blocking antibodies, PD-L1 blocking antibodies, LAG-3 blocking antibodies, Tim-3 blocking antibodies, TIGIT blocking antibodies, VISTA blocking antibodies, Siglec-15 blocking antibodies and combinations thereof;

Preferably, the vector is a virus;

More preferably, the virus is selected from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, poxviruses and herpesviruses.

5. An immune cell expressing the chimeric receptor of claim 1 or 2;

Preferably, the immune cells are selected from T cells, natural killer cells (NK), innate lymphoid cells (ILC), hematopoietic stem cells, embryonic stem cells and pluripotent stem cells;

More preferably, the T cells are selected from unsorted and purified T cells, sorted and purified T cells and natural killer T cells (NKT);

Further preferably, the sorted and purified T cells are selected from sorted and purified PD-1 ⁺ T cells, sorted and purified CD137 ⁺ T cells, sorted and purified CD160 ⁺ T cells, sorted and purified naive T cells ( T _naive ), sort-purified central memory T cells (T _CM ), sort-purified effector memory T cells ( _TEM ), sort-purified effector T cells ( _TEMRA ), sort-purified transitional memory cells T cells (Transitional Memory T cells, T _TM ) and sorted and purified tissue memory T cells (Tissueresidential memory T cells, T _RM ).

6. A combination comprising the immune cell and tumor antigen-targeting antibody or viral antigen-targeting antibody of claim 5;

Preferably, the tumor antigen is selected from one or more of the following: CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD70, CD117, CD123, CD133, CD138, CD147, CD171, NKG2DL, HER2 , MUC1, MUC16, CEA, EpCAM, IL-13Rα2, EGFR, EGFRvIII, GD2, DR5, EphA2, FRα, PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL , ROR1, ROR2, FAP, Mesothelin, DLL3 and CLDN18;

More preferably, the tumor antigen is selected from one or more of the following: HER2, EGFR, CD47, AXL and FAP;

Preferably, the viral antigen is selected from one or more of the following: gp120 of human acquired immunodeficiency virus HIV-1, surface antigen of hepatitis B virus HBV, hemagglutinin or neuraminidase of influenza virus, Ebola virus spike protein, severe acute respiratory syndrome coronavirus SARS-CoV surface spike protein, Middle East respiratory syndrome coronavirus MERS-CoV surface spike protein and novel coronavirus SARS-CoV-2 surface spike protein;

7. The combination of the chimeric receptor according to claim 1 or 2, the immune cell according to claim 5, the immune cell according to claim 6 and a tumor antigen-targeting antibody or a virus antigen-targeting antibody is used in preparation Use in drugs for the treatment of tumors or viral infectious diseases;

Preferably, the tumor is selected from one or more of the following: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasal cancer Pharyngeal cancer, small bowel cancer, colorectal cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, rhabdomyomas and leiomyomas;

Preferably, the viral infectious disease is selected from one or more of the following: Human Acquired Immunodeficiency Syndrome, Hepatitis B, Influenza, Ebola Virus Disease, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.

8. The preparation method of immune cells as claimed in claim 5, wherein the method comprises the steps:

1) obtaining the nucleic acid sequence of the chimeric receptor as claimed in claim 1 or 2;

2) cloning the nucleic acid sequence of the chimeric receptor into a lentiviral expression vector to obtain a lentiviral expression plasmid encoding the chimeric receptor;

3) Co-transfecting the lentiviral expression plasmid, backbone plasmid and envelope plasmid into HEK293T cells, packaging and obtaining lentiviral particles, and centrifuging and concentrating to obtain a lentiviral concentrate;

4) Transduce the lentivirus into immune cells to obtain immune cells expressing chimeric receptors;

Preferably, the immune cells are T cells.