Fusion protein of bispecific antibody targeting T cell and Her2 in series connection with IL-15 and receptor thereof and application of fusion protein

Abstract

The invention discloses a fusion protein of a bispecific antibody targeting a T cell and Her2 in series connection with IL-15 and a receptor thereof and an application of the fusion protein. Belongs to the technical field of biology. The fusion protein disclosed by the invention can be combined with a recombinant human CD3 protein and a recombinant human Her2 protein, can be combined with NK92 and SK-BR-3 cell lines, can stimulate NK92 cell proliferation, and can respectively cause ADCC effects mediated by T and NK cells; the antibody has important economic and social significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and more particularly to a fusion protein of a bispecific antibody targeting T cells and Her2 and a tandem IL-15 and its receptor and application thereof. BACKGROUND

[0002] Tumor immunotherapy is a treatment method that actively or passively produces tumor-specific immune responses in the body to inhibit and kill tumor cells, and has the advantages of high specificity and efficiency, and no harm to the body. Molecular targeted therapy and immune factor therapy are important components of immunotherapy.

[0003] Her2 overexpression exists in 15-20% of breast cancer patients, with a detection rate of about 2.5% in lung cancer, about 20% in gastric cancer, about 20% in cholangiocarcinoma, about 27% in ovarian cancer, and about 18-80% in endometrial cancer, and is associated with more aggressive disease and worse prognosis. Her2-targeted therapy has significantly improved the prognosis of Her2-positive breast cancer patients and gastric cancer patients. Currently, several anti-Her2 targeted drugs have been approved for the treatment of breast cancer: Trastuzumab and Pertuzumab, both of which are anti-Her2 human monoclonal antibodies. In the metastatic setting, patients can relapse after treatment. In the era of the continuous development of immunotherapy drugs, the understanding of the immune response to anti-HER2 antibodies is rapidly developing. Trastuzumab treatment can promote the activation of NK cells in breast cancer patients overexpressing Her2, indicating that the efficacy of Trastuzumab monotherapy may be related to antibody-dependent cell-mediated cytotoxicity (ADCC) in addition to direct targeting of Her2. In patients with cancer recurrence or progression, ADCC can be hindered by various factors, such as FcγRIIIa polymorphism and immunosuppressive microenvironment. Therefore, new drug development strategies are being studied to enhance the ADCC effect induced by anti-Her2 therapy.

[0004] Bispecific antibodies can bind two epitopes of the same antigen or two different antigopes due to the presence of two specificities of antibodies. Bispecific antibodies exert their functional properties through effector and target sites: the effector site serves to connect the recruitment of effector cells, drug molecules or viruses; the target site can target molecules or cells, etc. T cell bispecific antibodies are therapeutic antibodies that can connect T cells with tumor cells, and the design aims to direct T cells to tumor cells, so that T cells can effectively kill tumor cells. Most T cell engagers (TCEs) have three domains: one domain binds to a component of the T cell receptor, one domain binds to a tumor-associated antigen, and the third domain provides additional functions, such as extending half-life. Compared with conventional IgG, TCE is considered to be more effective than Fc-mediated ADCC. Compared with antibody drug conjugates (ADCs), the cytotoxicity of TCE depends on the host's immune system rather than the cytotoxicity of chemical payloads, and it attacks dormant and actively dividing cancer cells, which is safer, and has become a research hotspot of bispecific antibodies in recent years.

[0005] CD3 is an important accessory protein of T cell receptor (TCR), which generates downstream signals together with TCR. Traditional tumor antibody drugs mainly target TAA (tumor-associated antigen), which is highly expressed on tumor cells but has low expression level on healthy cells. CD3 kills tumors by T cells, not traditional TAA. CD3 drugs are mostly in the form of polyclonal antibodies, and are mainly designed in the form of CD3 combined with TAA. The TAA end helps the antibody target tumor tissue, and the CD3 end targets T cells, which release tumor-killing substances (perforin / granzyme, etc.) by T cells to inhibit tumors.

[0006] The aforementioned HER2 belongs to the tyrosine kinase receptor family, and there are more than 29 CD3 / HER2 bispecific antibodies in the clinical research stage. Among them, 11 products have entered the clinical I / II phase. With the mature understanding of the mechanism of CD3 and the gradual exploration of TAA, TCE drugs are expected to show more potential. As a new mechanism of tumor immunotherapy drugs, TCE drugs have gradually established themselves in hematological tumors, and are breaking through to solid tumors and autoimmune indications.

[0007] IL-15 belongs to the common gamma chain family factor member. Its alpha receptor is mainly expressed on the surface of antigen presenting cells, and IL-15 is also mainly secreted by these cells. Secreted IL-15 first binds to the IL-15Ralpha on the surface of antigen presenting cells (mononuclear, macrophage, DC cell and B cell, etc.), which on one hand increases the stability of IL-15, and on the other hand significantly improves the activity of IL-15. Subsequently, IL-15 combined with IL-15Ralpha binds to IL-15Rbeta through the surface of T cells or NK cells, plays a bridging or transactivation role, and establishes direct contact between T cells or NK cells and antigen presenting cells. IL-15 binds to IL15Rbeta to activate T cell and / or NK cell proliferation and provide growth factor signals. At the same time, by directly connecting antigen presenting cells and T cells, it also provides T cells with auxiliary activation signals and tumor antigen signals. In addition, IL-15 can induce NKG2D expression, which can effectively promote the expansion of effector and memory CTL cells even in the absence of CD28 signals. Through the interaction of IL-15-IL-15R, three key signals can be directly provided to T cells: tumor antigen signal, auxiliary activation signal and cytokine proliferation signal. In theory, the effectiveness of IL-15 in treating tumors (about 60%) is much higher than that of PD1 antibody and other immune checkpoint inhibitors (about 20%). In 2024, IL-15 was approved for marketing for the treatment of bladder cancer, with a complete remission rate of 60%~70%. IL-15 combined with PD1 antibody provides four signals to killer immune cells at the same time, so it is the best partner for immunotherapy. The effect of its combination therapy with other monoclonal antibodies needs to be further explored.

[0008] Therefore, how to provide a bispecific antibody targeting T cells and Her2 positive tumor cells and a fusion protein of the bispecific antibody and IL-15 and a preparation method and application thereof are technical problems to be solved by those skilled in the art. SUMMARY

[0009] Therefore, the present application provides a bispecific antibody targeting T cells and Her2 and a fusion protein of the bispecific antibody and IL-15 and its receptor and application thereof. The fusion protein is a fusion protein of a bispecific antibody and an active cytokine, which forms a heterodimer by two amino acid sequences and forms a tetramer by the disulfide bond of Fc; the fusion protein provides a new method for the detection, prevention or treatment of tumors, and has important economic and social significance.

[0010] In order to solve the above technical problems, the present application adopts the following technical solutions:

[0011] A fusion protein of a bispecific antibody targeting T cells and Her2 and IL-15 and its receptor, comprising a single chain variable region of a Her2 targeting antibody and a single chain variable region of a CD3 targeting antibody;

[0012] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the Her2 targeting antibody are shown as SEQ ID NO. 13~SEQ ID NO. 15;

[0013] GFNIKDTY, SEQ ID NO. 13;

[0014] IYPTNGYT, SEQ ID NO. 14;

[0015] SRWGGDGFYAMDY, SEQ ID NO. 15;

[0016] The amino acid sequences of CDR-L1, CDR-L2, CDR-L3 of the Her2 targeting antibody are shown as SEQ ID NO. 20, SAS, SEQ ID NO. 21;

[0017] QDVNTA, SEQ ID NO. 20;

[0018] QQHYTTPPT, SEQ ID NO. 21;

[0019] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the CD3 targeting antibody are shown as SEQ ID NO. 26~SEQ ID NO. 28;

[0020] GFTFNTYA, SEQ ID NO. 26;

[0021] IRSKYNNYAT, SEQ ID NO. 27;

[0022] ARHGNFGNSYVSWFAY, SEQ ID NO. 28;

[0023] The amino acid sequences of CDR-L1, CDR-L2, CDR-L3 of the CD3 targeting antibody are shown as SEQ ID NO. 33, GTN, SEQ ID NO. 34;

[0024] TGAVTTSNY, SEQ ID NO. 33;

[0025] ALWYSNLWV, SEQ ID NO. 34.

[0026] Further, the single chain variable region of the Her2 targeting antibody and the single chain variable region of the CD3 targeting antibody further comprise a framework region, and the amino acid sequences thereof are as follows:

[0027] The amino acid sequences of FR-H1, FR-H2, FR-H3, FR-H4 of the Her2 targeting antibody are shown in SEQ ID NO. 9~SEQ ID NO. 12;

[0028] EVQLVESGGGLVQPGGSLRLSCAAS, SEQ ID NO. 9;

[0029] IHWVRQAPGKGLEWVAR, SEQ ID NO. 10;

[0030] RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC, SEQ ID NO. 11;

[0031] WGQGTLVTVSS, SEQ ID NO. 12;

[0032] The amino acid sequences of FR-L1, FR-L2, FR-L3, FR-L4 of the Her2 targeting antibody are shown in SEQ ID NO. 16~SEQ ID NO. 19;

[0033] DIQMTQSPSSLSASVGDRVTITCRAS, SEQ ID NO. 16;

[0034] VAWYQQKPGKAPKLLIY, SEQ ID NO. 17;

[0035] FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC, SEQ ID NO. 18;

[0036] FGQGTKVEIK, SEQ ID NO. 19;

[0037] The amino acid sequences of FR-H1, FR-H2, FR-H3, FR-H4 of the CD3 targeting antibody are shown in SEQ ID NO. 22~SEQ ID NO. 25;

[0038] EVQLVESGGGLVQPGGSLRLSCAAS, SEQ ID NO. 22;

[0039] MNWVRQAPGKGLEWVAR, SEQ ID NO. 23;

[0040] YYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC, SEQ ID NO. 24;

[0041] WGQGTLVTVSS, SEQ ID NO. 25;

[0042] The amino acid sequences of FR-L1, FR-L2, FR-L3, FR-L4 of the CD3 targeting antibody are shown in SEQ ID NO. 29~SEQ ID NO. 32;

[0043] QTVVTQEPSLTVSPGGTVTLTCRSS, SEQ ID NO. 29;

[0044] ANWVQQKPGQAPRGLIG, SEQ ID NO. 30;

[0045] KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYC, SEQ ID NO. 31;

[0046] FGGGTKLTVL, SEQ ID NO. 32.

[0047] Further, the amino acid sequence of the heavy chain variable region of the Her2 targeting antibody is shown in SEQ ID NO. 5;

[0048] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, SEQ ID NO. 5;

[0049] The amino acid sequence of the light chain variable region of the Her2 targeting antibody is shown in SEQ ID NO. 6;

[0050] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, SEQ ID NO. 6;

[0051] The amino acid sequence of the heavy chain variable region of the CD3 targeting antibody is shown in SEQ ID NO. 7;

[0052] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS, SEQ ID NO. 7;

[0053] The amino acid sequence of the light chain variable region of the CD3 targeting antibody is shown as SEQ ID NO. 8;

[0054] QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL, SEQ ID NO. 8.

[0055] Further, the amino acid sequence of the single chain variable region of the Her2 targeting antibody is shown as SEQ ID NO. 3;

[0056] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, SEQ ID NO. 3;

[0057] The amino acid sequence of the single chain variable region of the CD3 targeting antibody is shown as SEQ ID NO. 4;

[0058] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL, SEQ ID NO. 4.

[0059] Further, IL-15 and its receptor IL-15Rα are also included;

[0060] The amino acid sequence of IL-15 is shown as SEQ ID NO. 35;

[0061] The amino acid sequence of IL-15 is shown as SEQ ID NO. 35;

[0062] The amino acid sequence of IL-15Rα is shown as SEQ ID NO. 36;

[0063] The amino acid sequence of IL-15Rα is shown as SEQ ID NO. 36;

[0064] Further, two amino acids are included, and the amino acid sequences are shown as SEQ ID NO. 1-SEQ ID NO. 2;

[0065] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD, SEQ ID NO. 1;

[0066] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLPSGQAGAAASESLFVSNHAYNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS, SEQ ID NO. 2.

[0067] Use of the fusion protein in preparation of reagents and / or drugs for detecting or treating Her2 positive tumor.

[0068] Through the technical solutions described above, compared with the prior art, the present application has the following beneficial effects:

[0069] (1) The fusion protein described in the present application contains Her2, CD3 targeting antibody, which can bind to Her2 expression positive target cells; can bind to CD3; contains IL-15, IL-5Rα segment, which can induce NK92 cell proliferation, has the potential to establish the connection between effector cells and antigen presenting cells; has ADCC activity on the basis of the above activities;

[0070] (2) The fusion protein described in the present application provides a new candidate for the detection and treatment of Her2 expression positive tumor, which has important economic and social significance. BRIEF DESCRIPTION OF DRAWINGS

[0071] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0072] Figure 1 It is the enzyme digestion identification result graph of the fusion protein expression plasmid in the embodiment 1 of the present application, wherein lane 1 is Marker, lane 2 is plasmid pcDNA3.4-30 band, lane 3 is plasmid pcDNA3.4-30 BamHI, EcoRI double enzyme digestion band, lane 4 is Marker, lane 5 is plasmid pcDNA3.4-148 band, lane 6 is plasmid pcDNA3.4-148 BamHI, EcoRI double enzyme digestion band;

[0073] Figure 2 It is the expression and purification result graph of the fusion protein prepared in the embodiment 1 of the present application, figure A is the corresponding graph of elution volume and OD 280 , pH; figure B is the SDS-PAGE result, lane 1 is Marker, lane 2 is 30 / 148 denatured and reduced sample band;

[0074] Figure 3 It is the binding ELISA result graph of the fusion protein prepared in the embodiment 2 of the present application and recombinant human CD3 protein;

[0075] Figure 4 It is the binding ELISA result graph of the fusion protein prepared in the embodiment 2 of the present application and recombinant human Her2 protein;

[0076] Figure 5 Binding FC results chart of the fusion protein prepared in Example 3 of the present application to the NK92 cell line and the human breast cancer cell line SK-BR-3;

[0077] Figure 6 Results chart of the fusion protein prepared in Example 4 of the present application inducing proliferation of NK92 cells;

[0078] Figure 7 Results chart of the fusion protein prepared in Example 5 of the present application inducing primary PBMC-derived T cells to kill SK-BR-3;

[0079] Figure 8 ADCC results chart of primary PBMC-induced NK cells killing SK-BR-3 mediated by the fusion protein prepared in Example 5 of the present application;

[0080] Figure 9 ADCC results chart of NK92 killing SK-BR-3 mediated by the fusion protein prepared in Example 5 of the present application. DETAILED DESCRIPTION

[0081] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0082] Example 1

[0083] Plasmid construction, protein expression and purification

[0084] (1) The aforementioned SEQ ID No. 1 (No. 148) and SEQ ID No. 2 (No. 30) sequences were synthesized by gene synthesis (restriction enzyme BamHI and EcoRI enzyme cutting sites were added at both ends of the sequences), and pcDNA3.4-TOPOTA cloning kit (purchased from Yingwei Jie (Shanghai) Trade Co., Ltd.) was used to construct pcDNA3.4 plasmid for expressing fusion protein, i.e. pcDNA3.4-148 and pcDNA3.4-30.

[0085] (2) The pcDNA3.4-148 and pcDNA3.4-30 plasmids of the previous step were transformed into E. coli TOP10, amplified by shaking culture in LB medium, extracted using a plasmid large extraction kit (Beijing Julongmei Biological Technology Co., Ltd.), and then subjected to enzyme cutting identification using restriction enzymes BamHI and EcoRI.Figure 1 ). From left to right lane, Marker, plasmid pcDNA3.4-30 band, plasmid pcDNA3.4-30 band of BamHI, EcoRI double enzyme digestion, Marker, plasmid pcDNA3.4-148 band, plasmid pcDNA3.4-148 band of BamHI, EcoRI double enzyme digestion, wherein the double enzyme digestion band is linearized plasmid band on the upper side of the band position, and the lower side is the target band position, which is consistent with the designed size position.

[0086] (3) The expression plasmid was transfected into 293F cells using PEI transfection reagent for protein expression.

[0087] (4) The supernatant was harvested 120 h after transfection, centrifuged and filtered, and then the protein A affinity column (purchased from Cytiva General Company) was treated with 5 times the column volume of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2), and the supernatant was loaded. After the end, the buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) was used to wash the impurities to the baseline; then the eluent 50 mM citric acid / sodium citrate buffer (pH 3.2) was used to elute the protein, and the sample with a UV absorbance of 100 mAu or more was collected; then the pH was adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), and after concentration with a concentration tube and sterilization by filtration, it was stored at 4°C. 3 μg of sample was taken for SDS-PAGE gel staining ( Figure 2 ): Figure 2 A is a chromatogram, which is a corresponding graph of elution volume and OD 280 , pH; Figure 2 B lane 1 is Marker, lane 2 is 30 / 148 denatured and reduced sample band, and the position of the two protein chains is consistent with the designed size position (30 / 148 forms a Y-shaped structure of the antibody after expression in cells, becoming a protein, and forms two bands under denatured and reduced conditions, Figure 2 B is displayed, wherein the upper one is the heavy chain, and the lower two light bands are the light chain, and part of them are glycosylated, so the light chain shows two bands), and the protein obtained at this step is the fusion protein 30 / 148.

[0088] Example 2

[0089] Antigen-antibody binding ELISA experiment

[0090] (1) Commercial recombinant human CD3 and Her2 proteins were diluted with pH 9.6 NaHCO3 coating solution, spread on ELISA plates, 100 ng / well, and incubated at 4°C overnight.

[0091] (2) Wash the ELISA plate coated yesterday with PBS for 3 times.

[0092] (3) Block with 3% BSA in PBST for 30 min at room temperature.

[0093] (4) Dilute the purified fusion protein with 3% BSA in PBST, start with 100 μg / mL, 10-fold dilution to 0.001 μg / mL, 6 concentrations in total, add to the ELISA plate, incubate for 30 min at room temperature.

[0094] (5) Wash with PBST for 3 times, add 100 μL mouse anti-human HRP secondary antibody (1:5000) per well, incubate for 30 min at room temperature.

[0095] (6) Wash with PBST for 3 times, add 50 μL TMB color developing solution per well, 5 min.

[0096] (7) Add 50 μL 1 M HCl per well, detect OD 450 absorbance value by microplate reader.

[0097] The results are shown in Figure 3 , Figure 4 .

[0098] From the results, the fusion protein has binding activity to both target proteins CD3 and Her2, and shows dose-dependent effect, compared with the control protein (negative control, Herceptin has no CD3 binding activity, 39 / 146 has no Her2 binding activity).

[0099] Example 3

[0100] Cell binding flow cytometry experiment

[0101] (1) Take 1×10 6 of NK92 cells and SK-BR-3 cells in logarithmic growth phase, centrifuge at 300 g for 5 min, discard the supernatant.

[0102] (2) Resuspend in 500 μL of normal saline, centrifuge, and wash twice.

[0103] (3) Add 50 μL of fusion protein at a concentration of 40 μg / mL, incubate at room temperature for 30 min.

[0104] (4) Centrifuge and discard the supernatant, resuspend in 500 μL of normal saline, centrifuge, and wash twice.

[0105] (5) Add 50 μL of diluted goat anti-human AF488 secondary antibody (2 μL / tube), incubate at room temperature for 30 min.

[0106] (6) Resuspend in 500 μL of normal saline, centrifuge, and wash twice.

[0107] (7) Resuspend in 500 μL of normal saline, and perform flow cytometry detection.

[0108] The results are shown in Table 1. Figure 5

[0109] The upper two graphs are the results of NK92 cells, and the lower two graphs are the results of SK-BR-3 cells. The left graph is the result of the control group, and the right graph is the result of the fusion protein experiment. The rightward shift indicates positive binding. According to the results, the fusion protein can bind to the NK92 and SK-BR-3 cells expressing antigens in situ, and has binding activity under physiological conditions.

[0110] Example 4

[0111] NK92 cell proliferation experiment

[0112] (1) Dilute the NK92 cells to 2 x 10 5 cells / mL, and plate 96-well plates at 100 μL / well. Incubate in an incubator overnight.

[0113] (2) Dilute the aforementioned purified fusion protein and IL-2 to 1 μg / mL using the culture medium.

[0114] (3) Discard the culture medium in the 96-well plate. According to the sample well arrangement, add blank culture medium to the control wells, add 1 μg / mL of IL-2 diluent to the IL-2 wells, and add 1 μg / mL of fusion protein 30 / 148 diluent to the experimental wells. Incubate in an incubator for 48 h.

[0115] (4) Add 10 μL of CCK-8 reagent to each well, and incubate in an incubator for 2 h.

[0116] (5) Detect the OD 450 of each well using a microplate reader.

[0117] The results are shown in Table 2. The difference between the IL-2 treatment group and the control group was significant (p = 0.0013), and the difference between the fusion protein 30 / 148 treatment group and the control group was significant (p = 0.0006). Figure 6 According to the results, the fusion protein can promote the proliferation of NK92 cells and has IL-15 activity.

[0118] Example 5

[0119] Antibody-dependent cell-mediated cytotoxicity experiment

[0120]

[0121] ​​(1) Adjust the density of SK-BR-3 (target cells) in logarithmic growth phase to 1 x 10 5 cells / mL after counting by digestion, and then place 50 μL / well, i.e. 5000 cells / well, in a 96-well plate and incubate in an incubator overnight.

[0122] (2) Dilute the aforementioned purified fusion protein and control protein 145 (control antibody, with ADCC effect on NK cells) with culture medium at an initial concentration of 10 μg / mL, and dilute to 0.1 μg / mL at a 10-fold concentration gradient, i.e. 10, 1, and 0.1 μg / mL.

[0123] (3) Discard the culture medium in the 96-well plate, and add 50 μL of the antibody diluted in the previous step, blank culture medium, etc. (see Tables 1-3) according to the sample well arrangement, and incubate in an incubator for 40 min to form a protein-target cell complex.

[0124] (4) Dilute the PBMC-induced T cells or NK cells or NK92 cells (effector cells) to 1 x 10 6 and / or 5 x 10 5 cells / mL (two ET effector-target ratios, i.e. 10:1 and 5:1, are set for T cell experiments; and an effector-target ratio of 10:1 is set for PBMC-NK and NK92 cell experiments), and add 50 μL of effector cells, blank culture medium, etc. according to the sample well arrangement, and incubate in an incubator for 5 h.

[0125] Table 1 Arrangement of PBMC-T against SK-BR-3

[0126] Background blank Effector spontaneous Target cell spontaneous 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 30 / 148-10 30 / 148-1 30 / 148-0.1

[0127] Note:

[0128] Background blank: contains culture medium; used to deduct the background absorbance value of low control and sample wells;

[0129] Effector spontaneous: contains culture medium and effector cells;

[0130] Lysis solution control: contains culture medium and 10 μL of Lysis Solution per well; used to deduct the background absorbance value of high control;

[0131] Target cell spontaneous: contains target cells and culture medium, without lysis treatment; used to determine the spontaneous LDH release of untreated normal cells;

[0132] Target cell lysis: contains target cells and culture medium, with 10 μL of Lysis Solution per well; used to determine the maximum releasable LDH of cells;

[0133] The same applies below.

[0134] Table 2 PBMC-NK against SK-BR-3

[0135] Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1

[0136] Table 3 NK92 against SK-BR-3

[0137] Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Background blank Effector spontaneous Target cell spontaneous 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1 Lysate control Effector spontaneous Target cell maximum 145-10 145-1 145-0.1 30 / 148-10 30 / 148-1 30 / 148-0.1

[0138] (5) After 4.5 h of the above step, 10 μL Lysis Solution in the LDH detection kit was added according to the setting of the sample well, and the incubation was continued to 5 h.

[0139] (6) The 96-well plate was taken out, 50 μL medium in each well was discarded, and then 50 μL Working Solution in the LDH detection kit was added to each well, and the incubation was carried out in the dark for 30 min.

[0140] (7) The aforementioned 96-well plate, Stop Solution in the LDH detection kit was added to each well, and the ABS: OD was detected by the enzyme label instrument. 490 .

[0141] (8) The cytotoxicity was calculated according to the cytotoxicity calculation formula.

[0142] ADCC experimental group = ABS (experimental group) - ABS (background blank);

[0143] Effector cell spontaneous release = ABS (effector spontaneous) - ABS (background blank);

[0144] Target cell spontaneous release = ABS (target cell spontaneous) - ABS (background blank);

[0145] Target cell maximum release = ABS (target cell maximum) - ABS (lysis solution control);

[0146] Cytotoxicity (%) = (ADCC experimental group - effector cell spontaneous release - target cell spontaneous release) / (target cell maximum release - target cell spontaneous release) x 100%.

[0147] The results are shown in Figure 7 , Figure 8 , Figure 9 .

[0148] It can be seen from the results that the fusion protein can cause antibody-dependent T cell and NK cell-mediated cytotoxicity, i.e. has ADCC activity.

[0149] The various embodiments described in this specification are intended to be illustrative only and in no way limit the scope of the application. Changes and modifications can be made by those skilled in the art, which employ the principles of the application, without departing from the scope thereof. Accordingly, the application is not limited to the embodiments described herein, but instead has scope to encompass any changes and modifications that serve the same, equivalent, or similar purposes.

[0150] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to those skilled in the art and are within the scope of the following claims. The description is presented in the best mode for making and using the application. What is claimed is:

Claims

1. A fusion protein targeting a bispecific antibody of T cells and Her2 and tandem IL-15 and its receptor, characterized in that, a single chain variable region of a Her2-targeting antibody and a single chain variable region of a CD3-targeting antibody; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the Her2-targeting antibody are shown as SEQ ID NO. 13~SEQ ID NO. 15; the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 of the Her2-targeting antibody are shown as SEQ ID NO. 20, SAS, SEQ ID NO. 21; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the CD3-targeting antibody are shown as SEQ ID NO. 26~SEQ ID NO. 28; the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 of the CD3-targeting antibody are shown as SEQ ID NO. 33, GTN, SEQ ID NO.

34.

2. The fusion protein of claim 1, wherein, the single chain variable region of the Her2-targeting antibody and the single chain variable region of the CD3-targeting antibody further comprise a framework region, and the amino acid sequences thereof are as follows: the amino acid sequences of FR-H1, FR-H2, FR-H3, FR-H4 of the Her2-targeting antibody are shown as SEQ ID NO. 9~SEQ ID NO. 12; the amino acid sequences of FR-L1, FR-L2, FR-L3, FR-L4 of the Her2-targeting antibody are shown as SEQ ID NO. 16~SEQ ID NO. 19; the amino acid sequences of FR-H1, FR-H2, FR-H3, FR-H4 of the CD3-targeting antibody are shown as SEQ ID NO. 22~SEQ ID NO. 25; the amino acid sequences of FR-L1, FR-L2, FR-L3, FR-L4 of the CD3-targeting antibody are shown as SEQ ID NO. 29~SEQ ID NO.

32.

3. The fusion protein of claim 1, wherein, the amino acid sequence of the heavy chain variable region of the Her2-targeting antibody is shown as SEQ ID NO. 5; the amino acid sequence of the light chain variable region of the Her2-targeting antibody is shown as SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region of the CD3-targeting antibody is shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region of the CD3-targeting antibody is shown as SEQ ID NO.

8.

4. The fusion protein of claim 1, wherein, the amino acid sequence of the single chain variable region of the Her2-targeting antibody is shown as SEQ ID NO. 3; the amino acid sequence of the single chain variable region of the CD3-targeting antibody is shown as SEQ ID NO.

4.

5. The fusion protein of claim 1, wherein, IL-15 and its receptor IL-15Rα are further included; the amino acid sequence of IL-15 is shown as SEQ ID NO. 35; the amino acid sequence of IL-15Rα is shown as SEQ ID NO.

36.

6. The fusion protein of claim 1, wherein, two amino acids are included, and the amino acid sequences are shown as SEQ ID NO. 1~SEQ ID NO.

2.

7. The fusion protein of any one of claims 1~6 for use in the preparation of a reagent and / or a medicament for detecting or treating Her2-positive tumors.

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