CN114853905B

CN114853905B - Scheme for treating tumors by combining genetically modified NK cells and antibodies

Info

Publication number: CN114853905B
Application number: CN202210390240.1A
Authority: CN
Inventors: 尹乐; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2022-11-22
Anticipated expiration: 2042-04-14
Also published as: WO2023197603A1; CN114853905A

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a scheme for treating tumors by combining genetically modified NK cells and antibodies. Specifically, the invention provides a fusion protein which comprises a membrane signal on a CD8a protein, a recognition functional region of a CD64 protein FC, a transmembrane structure and an intracellular signal transduction sequence of a CD16 protein and an intracellular signal transduction sequence of a 2B4 protein. Most preferably, NK cells expressing the fusion protein have a stronger killing activity.

Description

Scheme for treating tumors by combining genetically modified NK cells and antibodies

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a scheme for treating tumors by combining genetically modified NK cells and antibodies.

Background

Since the killing activity of NK cells (natural killer cells) is MHC-unrestricted, it is called natural killing activity. Unlike T cells and B cells, NK cells can recognize and kill target cells without specific antigen sensitization stimulation, and the killing effect of NK cells after acting on the target cells is early, and the killing effect can be seen in 1 hour in vitro and 4 hours in vivo.

Target cells of the NK cells mainly comprise certain tumor cells, virus infected cells, certain self tissue cells (such as blood cells), parasites and the like, so the NK cells are important immune factors for resisting tumors and infection of the body. The main mechanism of NK cell killing of target cells: 1. causing cell lysis by releasing perforin and granzyme; 2. inducing apoptosis of the target cell via a ligand-induced receptor-mediated apoptosis activation pathway; 3. releasing cytokines (including NK cell cytotoxic factor, NK cell tumor necrosis factor) to kill target cells; 4. antibody-dependent cell-mediated cytotoxicity (ADCC).

When an antibody binds to a tumor cell surface antigen via an antigen binding site and an Fc site binds to an Fc receptor on the surface of an immune effector cell, the immune effector cell is activated and kills the tumor cell, a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). There are three major Fc receptors: fc γ RI (CD 64), fc γ RII (CD 32), fc γ RIII (CD 16), wherein the latter two can be further classified as: fc γ RIIa, fc γ RIIb, fc γ RIIc, fc γ RIIIa, fc γ RIIIb. Different immune cells express specific Fc receptors, e.g., neutrophils typically express Fc γ RI, fc γ RII, fc γ RIIIb, whereas NK cells express only Fc γ RIIIa. Fc γ RIIIa is generally considered to be a key receptor for ADCC, so while NK cells, monocytes macrophages and neutrophils can all produce ADCC effects, NK cells are considered to be the most important cell population.

ADCC is dependent on many factors, such as the affinity of an antibody for an antigen, the affinity of an antibody for an Fc receptor, the density of tumor antigens, the characteristics of tumor target cells, and the characteristics of immune effector cells. In general, the tighter the bridging of tumor target cells to immune effector cells via antibodies, the stronger the ADCC effect. Thus, antibodies with high affinity for antigen or Fc receptors mediate stronger ADCC. Tumor cells expressing high levels of the target antigen are more sensitive to ADCC and are easily killed by ADCC.

The infiltration degree of NK cells in the tumor part also has an influence on the effect of immunotherapy, and the recruitment of NK cells into the tumor can effectively improve the anti-tumor immune response. Chemotactic factors and adhesion factors in a tumor microenvironment promote the increase of the infiltration degree of NK in tumor tissues by recruiting NK cells, so that an activating receptor on the surface of the NK cells can identify corresponding ligands on the surface of the tumor cells, and killing media such as perforin and the like are released to play a role in resisting tumor cytotoxicity.

NK cell therapy is a promising field of clinical research, and has been studied to verify its safety and primary efficacy for some cancer patients, and it will play an important role in future tumor immunotherapy. With the annual increase of the incidence of tumors, the search for a high-efficiency treatment method without toxic and side effects is a common effort direction for doctors and patients. The NK cell therapy can be used for treating various cancers independently or in combination with other treatment modes, and has extremely high application prospect.

Disclosure of Invention

In order to further improve the killing effect of the NK cells, the patent provides the genetically modified NK cells, and compared with the common NK cells, the genetically modified NK cells have stronger killing effect; further, when used in combination with an antibody, the genetically modified NK cell of the present invention recognizes the Fc-terminus of the antibody, and expresses a strong killing effect on tumor cells by the antibody recognizing a specific target.

In a first aspect, the present invention provides a fusion protein, which comprises a membrane signal on a CD8a (i.e., CD8 α) protein, a CD64 protein FC recognition functional region, a CD16 protein transmembrane structure and intracellular signal transduction sequence, and a 2B4 protein intracellular signal transduction sequence.

In one embodiment, the fusion protein consists of a membrane signal on the CD8a protein, a FC recognition functional region of the CD64 protein, a transmembrane structure and intracellular signaling sequence of the CD16 protein, and an intracellular signaling sequence of the 2B4 protein.

In one embodiment, the amino acid sequence of the membrane signal on the CD8a protein has 1, 2, 3, 4, 5 or more mutations from the sequence shown in SEQ ID No. 1 or as shown in SEQ ID No. 1.

In one embodiment, the amino acid sequence of the FC recognition functional region of the CD64 protein has 1, 2, 3, 4, 5 or more mutations from the sequence shown in SEQ ID No. 2, or as shown in SEQ ID No. 2.

In one embodiment, the amino acid sequence of the transmembrane structure and intracellular signaling sequence of the CD16 protein has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown in SEQ ID No. 3, or as shown in SEQ ID No. 3.

In one embodiment, the amino acid sequence of the 2B4 protein intracellular signaling sequence has 1, 2, 3, 4, 5 or more mutations from the sequence set forth in SEQ ID No. 4, or as set forth in SEQ ID No. 4.

In a most preferred embodiment, the amino acid sequence of the fusion protein, i.e., SEQ ID No. 1, 2, 3, 4, are linked in sequence.

In another aspect, the invention provides polynucleotides encoding the aforementioned fusion proteins.

In one embodiment, the nucleotide sequence of the membrane signal on the CD8a protein is as shown in SEQ ID No. 5.

In one embodiment, the nucleotide sequence of the FC recognition functional region of the CD64 protein is shown as SEQ ID No. 6.

In one embodiment, the nucleotide sequences of the transmembrane structure and intracellular signal transduction sequence of the CD16 protein are shown as SEQ ID No. 7.

In one embodiment, the nucleotide sequence of the intracellular signaling sequence of the 2B4 protein is as set forth in SEQ ID No. 8.

That is, most preferably, the polynucleotide provided by the present invention is composed of SEQ ID NO. 5, 6, 7, and 8 linked in sequence.

As used herein, the term "polynucleotide" includes a single nucleic acid as well as a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any nucleic acid type, such as DNA or RNA.

In another aspect, the present invention provides a vector comprising the aforementioned polynucleotide, or expressing the aforementioned fusion protein.

In one embodiment, the vector comprises a lentiviral vector (lentiviral expression vector), a retroviral vector (retroviral expression vector), an adenoviral vector (adenoviral expression vector).

As used in the examples of the present invention, the vector is a lentiviral vector, such as conventional vectors pRSV-Rev (Plasmid # 12253), pMDLg/pRRE (Plasmid # 12251), pMD2.G (Plasmid # 12259).

As used herein, the term "vector" can refer to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may include a nucleic acid sequence, such as an origin of replication, that permits it to replicate in a host cell. The vector may also include one or more selectable marker genes and other genetic elements known in the art. The particular type of vector contemplated herein may be associated with or incorporated into a virus to facilitate cell transformation.

In one embodiment, the vector is a mature, commercial vector or a vector that is self-constructed on demand.

In another aspect, the invention provides a genetically modified host cell comprising one or more of the fusion proteins, polynucleotides, vectors described above.

In one embodiment, the host cell is an autologous cell, or a heterologous cell, from the subject.

As used herein, the term "subject", "individual" or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

In one embodiment, the host cell comprises a stem cell, an immune cell. The immune cells include one or more of T cells, B cells, K cells, and NK cells.

In one embodiment, the immune cell is an NK cell.

Exemplary stem cells, as described herein, include undifferentiated or differentiated stem cells. The stem cell may be selected from the group consisting of mesenchymal stem cell, hematopoietic stem cell, embryonic stem cell and induced pluripotent stem cell.

In one embodiment, the stem cell is an Induced Pluripotent Stem Cell (iPSC), collectively referred to as induced pluripotent cells.

The host cell of the invention may also be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; CHO, COS, 293 cells, etc.

In another aspect, the invention provides pharmaceutical compositions comprising the aforementioned fusion proteins, polynucleotides, vectors, genetically modified host cells.

<xnotran> , , (matuximab, MORAb-009), , , , , , , , , , , , , , , , - , - , , , , , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , , . </xnotran>

The pharmaceutical composition can be prepared into tablets, capsules, granules, oral liquid, suspensions, injections, microspheres or liposomes with pharmaceutically acceptable excipients according to a conventional method.

The pharmaceutical compositions of the present invention may also comprise one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers, such as neutral buffered saline, and the like; a sulfate salt; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins, polypeptides or amino acids, such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and a preservative.

The "pharmaceutical composition" of the present invention refers to a chemical or biological composition suitable for administration to a mammal. Such compositions may be specifically formulated for administration via one or more of a number of routes including, but not limited to, oral, epidermal, epidural, inhalation, intraarterial, intracardiac, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, intraoral, parenteral, rectal, subcutaneous, subdermal, subcutaneous, transdermal and transmucosal.

In another aspect, the present invention provides a method for producing the aforementioned genetically modified host cell, the method comprising introducing one or more of the aforementioned fusion proteins, polynucleotides, vectors into a host cell, or the method comprising introducing one or more of the aforementioned fusion proteins, polynucleotides, vectors into a stem cell and then inducing differentiation of the stem cell into a host cell.

In one embodiment, the stem cell is an induced pluripotent stem cell.

In one embodiment, the induced pluripotent stem cell is a human-derived induced pluripotent stem cell that is self-prepared or a commercial induced pluripotent stem cell line.

In one embodiment, the host cell is an NK cell.

In one embodiment, the NK cells are derived from a self-produced human NK cell or a commercial NK cell line.

In one embodiment, the method of introduction comprises electroporation, lipofection, viral transfection.

In one embodiment, the method of introduction is viral transfection.

In one embodiment, the viral transfection comprises the use of a lentivirus or retrovirus.

In one embodiment, the viral transfection is with a retrovirus.

In another aspect, the invention provides the use of the aforementioned fusion proteins, polynucleotides, vectors, genetically modified host cells in the treatment of cancer or autoimmune diseases.

In another aspect, the invention provides the use of the aforementioned fusion protein, polynucleotide, vector, genetically modified host cell in the preparation of a medicament for treating cancer or an autoimmune disease.

In another aspect, the invention provides the use of the fusion protein, polynucleotide, vector, genetically modified host cell as described above in the preparation of a product for enhancing the therapeutic effect of an antibody.

More preferably, the use is in improving the effect of matuximab in treating prostate cancer.

In another aspect, the present invention provides a method of treatment comprising administering to a subject one or more of the foregoing fusion proteins, polynucleotides, vectors, genetically modified host cells.

The methods of the invention may be used in combination with other types of cancer treatment, such as chemotherapy, surgery, radiation therapy (radiotherapy), gene therapy, and the like.

Said chemotherapeutic treatment may be carried out using doxorubicin, vincristine, vinorelbine, paclitaxel, cisplatin, actinomycin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, epirubicin, etoposide, fluoroarabinoadenosine, in addition to the monoclonal antibody described above; fluorouracil, gemcitabine (Gemcitabine); herceptin, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, levophenylalanine mustard, mercaptopurine, methotrexate, mitomycin, mitoxantrone, dihydroxyanthrone, olanexine, procarbazine, tolazine, rituximab (Rituxan), steroids, streptozocin, streptozotocin (Taxotere, docetaxel), thioguanine, thiotepa, raltitrexed, topotecan, traquinovone, 5-fluorouracil, elodad (Xeloda), vinblastine, vindesine, vinorelbine, gleevec, hydroxycamptothecine and other therapeutic drugs.

The route of administration of the methods of the invention may be enteral or parenteral.

Preferably, the route of administration is parenteral, e.g., injection (intravenous, intramuscular, subcutaneous)

According to the present invention, a subject can be treated by injecting an effective dose of the genetically modified host cell of the present invention, and the infusion can be repeated several times until the desired therapeutic effect is achieved. The appropriate effective dose and number of injections will vary from patient to patient and will be determined by those skilled in the art based on the particular circumstances of the subject.

In one embodiment, the subject is a suspected or diagnosed cancer or autoimmune disease patient.

The term "cancer" as used herein encompasses any type of cancer, including solid and hematopoietic cancers.

Preferably, the cancer comprises a cancer derived from an organ selected from the group consisting of: peritoneum, liver, pancreas, lung, bladder, prostate, uterus, cervix, vagina, bone marrow, breast, skin, brain, lymph nodes, head and neck, stomach, intestine, colon, kidney, testis, and ovary.

Representative cancers well known to those of skill in the art include nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, renal carcinoma, cancer of the connective tissue, melanoma, lung carcinoma, intestinal carcinoma, colon carcinoma, rectal carcinoma, colorectal carcinoma, brain carcinoma, laryngeal carcinoma, oral carcinoma, liver carcinoma, bone carcinoma, pancreatic carcinoma, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, VHL disease, zollinger-elli syndrome, adrenal carcinoma, anal carcinoma, bile duct carcinoma, bladder carcinoma, ureter carcinoma, oligodendroglioma, neuroblastoma, meningioma, myeloma, osteochondroma, chondrosarcoma, ewing's sarcoma, primary unknown site cancer, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast carcinoma, paget's disease, cervical carcinoma, esophageal carcinoma, gallbladder carcinoma, head cancer, cervical carcinoma, and cervical carcinoma eye cancer, neck cancer, kidney cancer, wilms 'tumor, kaposi's sarcoma, prostate cancer, testicular cancer, hodgkin's disease, non-hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small bowel cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterus cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatin tumors, gum cancer, heart cancer, lip cancer, meningeal cancer, oral cancer, neural cancer, palate cancer, parotid cancer, peritoneal cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

Exemplary "autoimmune diseases" as described herein include, but are not limited to, addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, guillain-Barre syndrome, hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.

Drawings

FIG. 1 is a vector map of CA64162B4 according to the present invention.

FIG. 2 is a vector map of CD6416 of the control group according to the present invention.

FIG. 3 is a graph showing the results of identifying constructed NK cells expressing CA64162B 4.

Fig. 4 is a statistical result of tumor volume after treatment of a mouse model with NK cell-conjugated antibody expressing CA64162B 4.

FIG. 5 is a graph showing the results of tumor detection in mice treated with the NK cell-conjugated antibody for CA64162B4 expression in mice models.

FIG. 6 is a graph showing the results of staining tumor tissue sections after NK cell-associated antibody treatment for CA64162B 4.

Detailed Description

The present invention is further described with reference to the following examples, which are intended to be illustrative of the preferred embodiments of the invention only, and not to be limiting of the invention in any way. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 Effect verification of fusion protein provided by the present invention

1. Experimental materials:

293FT cells (ATCC, CRL-3249), DMEM medium (Hyclone, SH 30022.01), FBS (CellMax Total Bovine Serum, SA 101.02)

Conventional vectors pRSV-Rev (Plasmid # 12253), pMDLg/pRRE (Plasmid # 12251), pMD2.G (Plasmid # 12259), pLenti-EF 1. Alpha. -CA64162B4-Puro

2. Experimental methods and results

Construction of vectors

As shown in FIG. 1, the CD8a protein upper membrane signal (SEQ ID No.: 5), the CD64 protein FC recognition functional region (SEQ ID No.: 6), the CD16 protein transmembrane mechanism and intracellular signal transduction sequence (SEQ ID No.: 7) and the 2B4 protein intracellular signal transduction sequence (SEQ ID No.: 8) are integrated to form a nucleic acid sequence encoding a fusion protein, and the fusion protein sequence expressing the invention is connected into a lentiviral vector for packaging lentivirus and carrying out gene modification on NK cells. The obtained NK cells (designated as CA64162B4-NK, namely NK cells expressing the fusion protein provided by the invention) can be used together with antibodies for treating tumors.

As shown in FIG. 2, the vector expresses a fusion protein consisting of a CD64 protein FC recognition functional region (SEQ ID No.: 6), a CD16 protein transmembrane mechanism and an intracellular signal transduction sequence (SEQ ID No.: 7), which is named as CD6416.

Verification of successful preparation of NK cells

The method comprises the following steps:

1. 293T cells were transfected with CA64162B4 backbone vector and helper vector pMD2.G (addgen # 12259), pMDLg/pRRE (addgen # 12251), pRSV-Rev (addgen # 12253) in 10. Collecting supernatant 48 hours and 72 hours after transfection, and obtaining lentivirus after purification and concentration;

2. mixing the concentrated lentivirus with purified lentivirus of NK92MI cells at 200 μ L per 100 ten thousand cells, and then placing in a 37 ℃ incubator 5% ₂ Carrying out conditional culture, and completely changing the culture solution after 24 hours;

3. culturing the cells obtained in the step 2 for 3 days, screening by using puromycin, then diluting and paving in a 96-well plate in a gradient manner, and performing monoclonal culture;

4. the single clones in 3 were subjected to flow assay (results are shown in FIG. 3).

In vivo killing activity verification

Mice were inoculated subcutaneously with C42-EGFP cells (at 500 ten thousand cells/mouse) on day-14, tail vein reinfusion on day 1, and DPBS (200 μ l/mouse) reinfused in control groups; experimental group 1 reinfused antibody matuximab (MORAB-009) (20 mg/mouse); experimental group 2 reinfused NK-CD6416 (500 ten thousand cells/mouse) + antibody matuximab (MORAB-009) (20 mg/mouse); experimental group 3 infusion of NK-CA64162B4 (500 ten thousand cells/mouse) + antibody matuximab (MORAB-009) (20 mg/mouse). Mice were imaged in vivo on days 0, 7, and 14, and tumor sizes were observed.

The statistical results of the volume are shown in FIG. 4, the results of the mouse living body imaging are shown in FIG. 5, and the obvious combination of NK-CA64162B4 and the antibody has better effect.

On day 0, tumor tissue section staining observation was performed 4 hours and 16 hours after tail vein reinfusion, as shown in fig. 6, the orange part (bubble shape) of the experimental group 2 was NK-CA64162B4 cells (human CD56 staining), infiltration of NK-CA64162B4 cells in tumor tissue and killing of tumor cells were observed at 4 hours after tail vein reinfusion, and a cavity caused by tumor cell death was observed at 16 hours. Compared with the antibody which is independently returned (experiment group 1), the NK-CA64162B4 cell and the antibody are combined (experiment group 2), the effect is obviously better.

Namely, the NK-CA64162B4 provided by the invention has the effect of improving the therapeutic effect of the antibody.

Sequence listing

<110> Shino Rev medicine science and technology (New zone of Zhuhai horizontal organ) Co., ltd

<120> a scheme for treating tumor by using genetically modified NK cell in combination with antibody

<141> 2022-04-12

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 1

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

20

<210> 2

<211> 274

<212> PRT

<213> Artificial Sequence

<400> 2

Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser Val Phe

1 5 10 15

Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu Pro Gly

20 25 30

Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln Thr Ser

35 40 45

Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser Gly Glu

50 55 60

Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile Gln Leu

65 70 75 80

Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg Val Phe

85 90 95

Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys Asp Lys

100 105 110

Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe Lys Phe

115 120 125

Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile Ser His

130 135 140

Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr Thr Ser

145 150 155 160

Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro Val Leu

165 170 175

Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val Thr Leu

180 185 190

Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu Tyr

195 200 205

Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn Thr Ser

210 215 220

Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly Leu Tyr

225 230 235 240

Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg Ser Pro

245 250 255

Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val Trp

260 265 270

Phe His

<210> 3

<211> 46

<212> PRT

<213> Artificial Sequence

<400> 3

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

1 5 10 15

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

20 25 30

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

35 40 45

<210> 4

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 4

Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu

1 5 10 15

Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn

20 25 30

His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser

35 40 45

Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr

50 55 60

Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys

65 70 75 80

Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly

85 90 95

Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu

100 105 110

Leu Glu Asn Phe Asp Val Tyr Ser

115 120

<210> 5

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 5

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 6

<211> 822

<212> DNA

<213> Artificial Sequence

<400> 6

accacaaagg cagtgatcac tttgcagcct ccatgggtca gcgtgttcca agaggaaacc 60

gtaaccttgc actgtgaggt gctccatctg cctgggagca gctctacaca gtggtttctc 120

aatggcacag ccactcagac ctcgaccccc agctacagaa tcacctctgc cagtgtcaat 180

gacagtggtg aatacaggtg ccagagaggt ctctcagggc gaagtgaccc catacagctg 240

gaaatccaca gaggctggct actactgcag gtctccagca gagtcttcac ggaaggagaa 300

cctctggcct tgaggtgtca tgcgtggaag gataagctgg tgtacaatgt gctttactat 360

cgaaatggca aagcctttaa gtttttccac tggaattcta acctcaccat tctgaaaacc 420

aacataagtc acaatggcac ctaccattgc tcaggcatgg gaaagcatcg ctacacatca 480

gcaggaatat ctgtcactgt gaaagagcta tttccagctc cagtgctgaa tgcatctgtg 540

acatccccac tcctggaggg gaatctggtc accctgagct gtgaaacaaa gttgctcttg 600

cagaggcctg gtttgcagct ttacttctcc ttctacatgg gcagcaagac cctgcgaggc 660

aggaacacat cctctgaata ccaaatacta actgctagaa gagaagactc tgggttatac 720

tggtgcgagg ctgccacaga ggatggaaat gtccttaagc gcagccctga gttggagctt 780

caagtgcttg gcctccagtt accaactcct gtctggtttc at 822

<210> 7

<211> 138

<212> DNA

<213> Artificial Sequence

<400> 7

gtctctttct gcttggtgat ggtactcctt tttgcagtgg acacaggact atatttctct 60

gtgaagacaa acattcgaag ctcaacaaga gactggaagg accataaatt taaatggaga 120

aaggaccctc aagacaaa 138

<210> 8

<211> 360

<212> DNA

<213> Artificial Sequence

<400> 8

tggaggagaa agaggaagga gaagcagtca gagactagtc ccaaggaatt tttgacaatt 60

tacgaagatg tcaaggatct gaaaaccagg agaaatcacg agcaggagca gacttttcct 120

ggagggggga gcaccatcta ctctatgatc cagtcccagt cttctgctcc cacgtcacaa 180

gaaccagcat atacattata ttcattaatt cagccttcca ggaagtctgg ttccaggaag 240

aggaaccaca gcccttcctt caatagcact atctatgaag tgattggaaa gagtcaacct 300

aaagcccaga accctgctcg attgagccgc aaagagctgg agaactttga tgtttattcc 360

Claims

1. A fusion protein, which consists of a CD8a protein upper membrane signal, a CD64 protein FC recognition functional region, a CD16 protein transmembrane structure and intracellular signal transduction sequence and a 2B4 protein intracellular signal transduction sequence,

the amino acid sequence of the membrane signal on the CD8a protein is shown as SEQ ID No. 1, the amino acid sequence of the FC recognition functional region of the CD64 protein is shown as SEQ ID No. 2, the amino acid sequences of the transmembrane structure and the intracellular signal transduction sequence of the CD16 protein are shown as SEQ ID No. 3, and the amino acid sequence of the intracellular signal transduction sequence of the 2B4 protein is shown as SEQ ID No. 4,

the amino acid sequence of the fusion protein is SEQ ID NO. 1, 2, 3 and 4 which are connected in sequence.

2. A polynucleotide encoding the fusion protein of claim 1.

3. The polynucleotide according to claim 2, wherein the fusion protein has the nucleotide sequence of the membrane signal of CD8a protein as shown in SEQ ID No. 5.

4. The polynucleotide of claim 2, wherein the fusion protein comprises a nucleotide sequence of a recognition functional region of CD64 protein FC as shown in SEQ ID No. 6.

5. The polynucleotide according to claim 2, wherein the fusion protein comprises the transmembrane structure of CD16 protein and the intracellular signal transduction sequence of the polynucleotide as shown in SEQ ID No. 7.

6. The polynucleotide of claim 2, wherein the nucleotide sequence of the intracellular signal transduction sequence of the 2B4 protein in the fusion protein is shown in SEQ ID No. 8.

7. The polynucleotide of claim 2, wherein the polynucleotide is composed of SEQ ID No. 5, 6, 7, and 8 linked in sequence.

8. Expressing the fusion protein of claim 1 or a vector comprising the polynucleotide of claim 2.

9. The vector of claim 8, wherein the vector comprises a lentiviral vector, a retroviral vector, or an adenoviral vector.

10. A genetically modified host cell comprising or expressing therein one or more of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 8.

11. The host cell of claim 10, which is an autologous cell or an allogeneic cell from a subject.

12. The host cell of claim 11, wherein the subject comprises a human, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, bear.

13. The host cell of claim 10, wherein the host cell comprises a stem cell or an immune cell.

14. The host cell of claim 13, wherein the immune cell comprises one or more of a T cell, a B cell, a K cell, and an NK cell.

15. The host cell of claim 14, wherein the immune cell is an NK cell.

16. The host cell of claim 13, wherein the stem cell is an induced pluripotent stem cell.

17. A pharmaceutical composition comprising one or more of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 8, and the host cell of claim 10.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is in the form of tablet, capsule, granule, oral liquid, suspension, injection, microsphere or liposome.

19. The pharmaceutical composition of claim 17, which may further comprise one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

20. The pharmaceutical composition of claim 17, further comprising a monoclonal antibody.

21. The pharmaceutical composition according to claim 20, wherein the pharmaceutically acceptable salt thereof, the monoclonal antibody comprises matuzumab, trastuzumab, cetuximab, daclizumab, ranibizumab, abamectin, adalimumab, alfuzumab, alemtuzumab, pemphidizumab, amazetuzumab, aprezumab, bazedozumab, betuzumab, belimumab, bevacizumab, motuzumab, slemtuzumab, belunumab-veduloxetine, mocratizumab, lattuzumab, carpuzumab, pentostatin, cetuzumab, canaumumab, and daclizumab, daluzumab, deluzumab, eimecoximab, efecolomab, elobizumab, ensliximab, epratuzumab, elmastomab, edazumab, faruzumab, phenotuzumab, galiximab, gemtuzumab ozogamicin, gelitumumab-vedoline, ibritumomab, agozemab, laneven mab, infliximab, influzumab ozotacin, ipilimumab, itumumab, labuzumab, lebevacizumab, lexazumab, lintuzumab, molovizumab.

22. A method for producing a host cell with high killing activity in vitro, the method comprising any one of:

1) Introducing one or more of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 8 into a host cell;

or the like, or, alternatively,

2) One or more of the fusion protein of claim 1, the polynucleotide of claim 2, and the vector of claim 8 is introduced into a stem cell, and the stem cell is then induced to differentiate into a host cell.

23. The method of claim 22, wherein the stem cell is an induced pluripotent stem cell.

24. The method of claim 23, wherein the induced pluripotent stem cell is derived from a self-prepared induced pluripotent stem cell of human origin or is a commercial induced pluripotent stem cell line.

25. The method of claim 22, wherein the host cell is an NK cell.

26. The method of claim 25, wherein the NK cells are derived from a self-produced human NK cell or a commercial NK cell line.

27. The method of claim 26, wherein the commercial NK cell line is NK92MI cells.

28. The method of claim 22, wherein the method of introduction comprises electroporation, lipofection, viral transfection.

29. The method of claim 28, wherein the introducing is viral transfection.

30. The method of claim 29, wherein the viral transfection comprises use of a lentivirus or retrovirus.

31. The method of claim 30, wherein the viral transfection is performed using a retrovirus.

32. Use of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 8, the host cell of claim 10, or the pharmaceutical composition of claim 17 for the preparation of a medicament for the treatment of cancer.

33. The use of claim 32, wherein the cancer comprises a cancer derived from an organ selected from the group consisting of: peritoneum, liver, pancreas, lung, bladder, prostate, uterus, cervix, vagina, bone marrow, breast, skin, brain, lymph nodes, head and neck, stomach, intestine, colon, kidney, testis, and ovary.

34. <xnotran> 32 , , , , , , , , , , , , , , , , , , , , , , T / , , VHL , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . </xnotran>

35. Use of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 8, the host cell of claim 10, or the pharmaceutical composition of claim 17 for the manufacture of a product for enhancing the therapeutic effect of a monoclonal antibody.

36. The use according to claim 35, wherein, the monoclonal antibodies include matuzumab, trastuzumab, cetuximab, daclizumab, ranibizumab, abamectin, alemtuzumab, pemphidizumab, ametuzumab pegol, alemtuzumab, apraxizumab, bravazumab, betuzumab, belimumab, bevacizumab, motaltuzumab, lactuzumab, carpuzumab pentapeptide, rituximab, poisesituzumab, cetuximab, cotuzumab, and rituximab daclizumab, daluzumab, gemtuzumab, eimexib, edeluzumab, ilolizumab, ensliximab, epratuzumab, elmatosumab, efletuzumab, phentuzumab, galiximab, gemtuzumab ozogamicin, gemtuximab, gelitumumab-velutin, ibritumomab tiuxetan, agozeumab, laredacimab, inflitumumab, ozomicin, ipilimumab, itumumab, rabuzumab, lexezumab, lintuzumab, and molovizumab.

37. The use of claim 35, wherein the antibody is matuximab.

38. The use of claim 35, wherein the therapeutic effect is an effect of treating prostate cancer.