CN112646043A

CN112646043A - Recombinant bifunctional fusion protein and application thereof

Info

Publication number: CN112646043A
Application number: CN202011559808.5A
Authority: CN
Inventors: 姜东成; 王芳; 杜宜彬; 陈鹏; 刘志锋
Original assignee: Hebei Shenyu Biotechnology Co ltd; Nanjing Huayan Biotechnology Co ltd
Current assignee: Hebei Shenyu Biotechnology Co ltd; Nanjing Huayan Biotechnology Co ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-13
Anticipated expiration: 2040-12-25
Also published as: CN112646043B

Abstract

The invention provides a recombinant bifunctional fusion protein and application thereof, and relates to the technical field of fusion proteins. The fusion protein is formed by connecting an anti-human MSLN antibody and a single domain structure combined with human CD47 protein. The protein can be combined with MSLN and CD47 at the same time, thereby playing a role in treating MSLN positive tumor cells through antibody-dependent, cell-mediated cytotoxicity and complement-dependent cytotoxicity activities, blocking the combination of CD47 and SIRP on the surfaces of macrophages, activating the phagocytosis of the macrophages to the tumor cells, having remarkable anti-tumor activity, having a tumor inhibition effect superior to that of single anti-MSLN monoclonal antibody treatment, and being capable of being used for treating tumors which are ineffective or tolerant to the anti-MSLN monoclonal antibody.

Description

Recombinant bifunctional fusion protein and application thereof

Technical Field

The invention belongs to the technical field of fusion proteins, and particularly relates to a recombinant bifunctional fusion protein and application thereof.

Background

Mesothelin (MSLN) is a 40kDa cell surface glycoprotein, is not a cancer-specific antigen, is a differential antigen, and is expressed at relatively low levels in normal tissues. Mesothelin is highly expressed in various types of solid tumors, such as pancreatic cancer, ovarian cancer, malignant mesothelioma, lung adenocarcinoma, and endometrial, biliary tract, gastric, and prostate cancers, as compared to normal tissue. Mesothelin is expressed on normal mesothelial cells lining the pleura, pericardium and peritoneum. The limited distribution of mesothelin in normal tissues makes it a promising target for tumor-specific therapy. The mesothelin gene encodes a 71kDa precursor protein, encoded by a 2138bp long cDNA, consisting of 628 amino acids, which is processed into a 40kDa membrane-associated protein (called mesothelin) and a 31kDa abscission fragment, called megakaryocyte-enhancing factor (MPF), which is released from the cell. Mesothelin protein is an antigen recognized by monoclonal antibody K1, and is typically a protein linked to membrane-bound glycolipid inositol (GPI); whereas MPF was isolated from the culture medium of a human pancreatic cancer cell line. Mesothelin is overexpressed in about 30% of cancers, and expression of tumor mesothelin is often associated with increased tumor invasiveness and clinically poor prognosis.

Signal-regulatory protein (SIRP) is a transmembrane glycoprotein and belongs to the family of transmembrane proteins. Signal-regulatory proteins have three members: SIRP alpha (CD172a), SIRP beta (CD172b), SIRP gamma (CD172 g). These three members have similar extracellular domains, but different intracellular domains. The extracellular domain comprises three immunoglobulin (Ig) -like regions, the first of which is an N-terminal V-like domain (D1), and the second and third of which is two C1-like domains. The intracellular domain of sirpa (CD172a) contains two inhibitory signaling regions that implicitly inhibit signaling and the corresponding cellular functions. SIRP is mainly expressed in macrophages (M), Dendritic Cells (DC) and neurons.

CD47 is also a penetrating glycoprotein belonging to the immunoglobulin superfamily and expressed on the surface of all cell types including erythrocytes, and has multiple functions in cell-to-cell communication. CD47 interacts with a variety of ligands, such as integrins, sirpa, SIRP γ, and thrombospondins. CD47 interacts with sirpa and CD47 down-regulates the activity of phagocytes expressing sirpa. It was found that many tumor cells over-express CD47 in addition to CD47 expressed in normal tissues and in this way, transmit a "do not eat me" signal, thereby avoiding killing by innate immune cells (immune escape) found by immune surveillance of the body. CD47 inhibits the maturation and activation of Dendritic Cells (DCs). CD47 is also involved in processes such as apoptosis, survival, proliferation, adhesion, migration, and regulation of angiogenesis, blood pressure, tissue perfusion, or platelet homeostasis. In cancers such as leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, and glioma, high levels of CD47 are associated with poor clinical outcomes. To date there is no bispecific antibody targeting both MSLN and CD 47.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant bifunctional fusion protein and its application, wherein the fusion protein simultaneously binds to MSLN and CD47, thereby exerting therapeutic effect on MSLN positive tumor cells, and blocking the binding of CD47 to SIRP on the surface of macrophages, stimulating the phagocytosis of tumor cells by macrophages, and having significant anti-tumor activity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a recombinant bifunctional fusion protein, which is formed by connecting an anti-human MSLN antibody and a single domain structure combined with human CD47 protein, and comprises a first combination structure domain and a second combination structure domain;

the first binding domain specifically binds to the target molecule human MSLN protein; the second binding domain specifically binds to the target molecule CD47 protein.

Preferably, the single domain structure that binds to human CD47 protein includes an extra-membrane N-terminal V-like domain derived from human signal-regulating protein (D1).

Preferably, the amino acid sequence of the N-terminal V-like domain (D1) derived from the outside of the human signal regulatory protein membrane is shown as SEQ ID NO. 1.

Preferably, the single domain structure that binds to human CD47 protein further comprises amino acids that have at least 80%, 85%, 90%, 95%, 98%, or 99% similarity to the sequence shown in SEQ ID No. 1.

Preferably, the fusion protein comprises a heavy chain comprising the polypeptide chain VH and a light chain_MSLN-CH1-Fc, the light chain comprising polypeptide chain V_SIRPαD1-(X)_n-VL_MSLN-CL; wherein VH_MSLNCH1 is the first constant domain of the heavy chain constant region of an anti-human MSLN antibody heavy chain; v_SIRPαD1An extra-membrane N-terminal V-like domain derived from human signal-regulating protein for paired binding to CD47 (D1); x is a linker, n is more than or equal to 0; VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

Preferably, said peptide chain VH_MSLNThe amino acid sequence of-CH 1-Fc is shown in SEQ ID NO. 2; the polypeptide chain V_SIRPαD1-(X)_n-VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 3.

Preferably, the fusion protein comprises a heavy chain comprising the polypeptide chain VH and a light chain_MSLN-CH1-Fc-(X)_n-V_SIRPαD1Said light chain comprising a polypeptide chain VL_MSLN-CL; wherein VH_MSLNA variable domain that is an anti-human MSLN antibody heavy chain; CH1 is the first constant domain of the heavy chain constant region; x is a linker, n is more than or equal to 0; v_SIRPαD1An extra-membrane N-terminal V-like domain derived from human signal-regulating protein for paired binding to CD47 (D1); VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

Preferably, said polypeptide chain VH_MSLN-CH1-Fc-(X)_n-V_SIRPαD1The amino acid sequence of (A) is shown in SEQ ID NO. 4; said polypeptide chain VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 5.

Preferably, the fusion protein comprises a heavy chain comprising polypeptide chain V and a light chain_SIRPαD1-(X)_n-VH_MSLN-CH1-Fc, the light chain comprising a polypeptide chain VL_MSLN-CL; wherein V_SIRPαD1An extra-membrane N-terminal V-like domain derived from human signal-regulating protein for paired binding to CD47 (D1); x is a linker, n is more than or equal to 0; VH_MSLNA variable domain that is an anti-human MSLN antibody heavy chain; CH1 is the first constant domain of the heavy chain constant region; VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

Preferably, the peptide chain V_SIRPαD1-(X)_n-VH_MSLNThe amino acid sequence of-CH 1-Fc is shown as SEQ ID NO.6, and the polypeptide chain VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 7.

Preferably, the domain source of the Fc comprises wild type or mutant; linker X is a polypeptide chain consisting of a plurality of identical or different amino acids.

The invention also provides a medicament for treating MSLN positive tumors, and the effective component of the medicament comprises the fusion protein.

The invention provides a recombinant dual-function fusion protein (MSLNmAb-V)_SIRPα) The expression vector is prepared by combining an anti-human MSLN (methamine, mesothelin) antibody with human Signal-regulatory protein α (Signal-regulatory proteins α, SIRP α, NCBI database: and Ig-like V regions at the outer ends of the AAH75849.1(Glu31-His164) membranes are connected. The protein can be combined with MSLN and CD47 at the same time, thereby playing a role in treating MSLN positive tumor cells through antibody-dependent and cell-mediated cytotoxic activity, blocking the combination of CD47 and SIRPa on the surfaces of macrophages, stimulating the phagocytosis of the macrophages to the tumor cells, having remarkable anti-tumor activity, having a tumor inhibition effect superior to that of single anti-MSLN monoclonal antibody treatment, and being capable of being used for treating patients with tumors which are ineffective or tolerant to the anti-MSLN monoclonal antibody.

Drawings

FIG. 1 is a schematic representation of the structure of a fusion protein; in FIG. 1, A is MSLN antigen; b is CD47 antigen;

FIG. 2 shows DOT detection protein expression;

FIG. 3 target binding activity assay, wherein FIG. 3A is target binding activity (MSLN) and FIG. 3B is target binding activity (human CD 47);

FIG. 4 shows the effect of HY202-1, HY202-2, and HY202-3 on ADCC effector cell activation activity, where FIG. 4A is the NCIH226 reporter gene assay and FIG. 4B is the OVCAR-3 reporter gene assay;

FIG. 5 shows the effect of HY202-1, HY202-2 and HY202-3 on macrophage phagocytic activity;

FIG. 6 shows the in vivo antitumor activities of HY202-1, HY202-2 and HY 202-3.

Detailed Description

The invention provides a recombinant dual-function fusion protein (MSLNmAb-SIRPa), which is formed by connecting an anti-human MSLN antibody and a single-domain structure combined with human CD47 protein, and comprises a first combination structure domain and a second combination structure domain;

The single domain structure of the human CD47 binding protein preferably comprises an extramembranous N-terminal V-like domain (D1) of a human signal regulatory protein (SIRPa), and the amino acid sequence of the extramembranous N-terminal V-like domain (D1) of the human signal regulatory protein is preferably shown as SEQ ID NO. 1. In the present invention, the single domain structure that binds to human CD47 protein preferably further comprises amino acids having at least 80%, 85%, 90%, 95%, 98% or 99% similarity to the sequence shown in SEQ ID No. 1.

The thawing protein of the invention has three different structures, the specific structural diagram is shown in figure 1, the fusion protein of the first structure comprises polypeptide chain VH_MSLN-CH1-Fc (heavy chain) and polypeptide chain V_SIRPαD1-(X)_n-VL_MSLN-CL (light chain); the fusion protein of the second structure comprises polypeptide chain VH_MSLN-CH1-Fc-(X)_n-V_SIRPαD1(heavy chain) and polypeptide chain VL_MSLN-CL (light chain); integration of the third StructureThe synthetic protein comprises polypeptide chain V_SIRPαD1-(X)_n-VH_MSLN-CH1-Fc (heavy chain) and polypeptide chain VL_MSLN-CL (light chain); wherein V_SIRPαD1An extra-membrane N-terminal V-like domain (D1) derived from human signal-regulating protein for binding to CD47, X is a linker, N is 0 or more, and VL_MSLNIs a variable domain of an anti-human MSLN antibody light chain, and CL is an anti-human MSLN antibody light chain constant domain; VH_MSLNCH1 is the first constant domain of the heavy chain constant region of an anti-human MSLN antibody heavy chain.

The peptide chain VH of the invention_MSLNThe amino acid sequence of-CH 1-Fc is preferably shown in SEQ ID NO. 2; the polypeptide chain V_SIRPαD1-(X)_n-VL_MSLNThe amino acid sequence of-CL is preferably as shown in SEQ ID NO. 3; said polypeptide chain VH_MSLN-CH1-Fc-(X)_n-V_SIRPαD1The amino acid sequence of (A) is preferably as shown in SEQ ID NO. 4; said polypeptide chain VL_MSLNThe amino acid sequence of-CL is preferably shown in SEQ ID NO. 5; the polypeptide chain V_SIRPαD1-(X)_n-VH_MSLNThe amino acid sequence of-CH 1-Fc is shown in SEQ ID NO. 6; said polypeptide chain VL_MSLNThe amino acid sequence of-CL is preferably shown in SEQ ID NO. 7.

The domain source of the Fc of the present invention preferably includes wild type or mutant type, more preferably wild type, and in the present embodiment, the amino acid sequence of the wild type Fc used is preferably as shown in SEQ ID No. 8. The linker X according to the invention is preferably a polypeptide chain consisting of a plurality of identical or different amino acids, and the amino acid sequence of the linker unit X in the examples according to the invention is preferably as shown in SEQ ID NO. 9.

In the recombinant dual-function fusion protein, a human MSLN antibody can kill MSLN positively expressed tumor cells, SIRP alpha can activate macrophages to phagocytose CD47 positively expressed tumor cells, and after the two are combined, the three different structures are obtained, and the tumor cells can be killed through two signal paths of MSLN and CD 47.

The preparation method of the fusion protein is not particularly limited, and the preferable method comprises the steps of artificially synthesizing nucleotide sequences for coding polypeptide chains of the fusion proteins and respectively cloning the nucleotide sequences to vectors, so as to respectively construct HY202-1, HY202-2 and HY202-3 expression vectors; and (3) electrically transferring CHO-K1 cells by using the expression vector, and inducing protein expression and purifying. The method for electrotransformation, induction of protein expression and purification is not particularly limited in the invention, and conventional technical means in the field can be utilized.

The invention also provides the application of the fusion protein in preparing a medicament for treating MSLN positive tumors. The fusion protein can be combined with MSLN and CD47 at the same time, thereby playing a role in treating MSLN positive tumor cells through antibody-dependent, cell-mediated cytotoxicity and complement-dependent cytotoxic activities, blocking the combination of CD47 and SIRP on the surface of macrophages, stimulating the phagocytosis of the macrophages on the tumor cells, and having significant anti-tumor activity; the tumor inhibition effect of the single anti-MSLN monoclonal antibody is better than that of single anti-MSLN monoclonal antibody; can be used for treating patients with tumors that are refractory or resistant to MSLN monoclonal antibody.

The following examples are provided to illustrate the present invention and the application of the present invention in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Expression vector construction

The antibody heavy chain adopts Amatuximab monoclonal antibody VH_MSLN-CH1-Fc (SEQ ID NO.10), the gene sequence was submitted to Nanjing Kinsley synthesis (item No.: C9962EE 050-2); will V_SIRPαD1(SEQ ID NO.11) and antihuman MSLN antibody light chain (SEQ ID NO.12) fusion, the gene sequence was delivered to Nanjing Kinsley synthesis (project No.: C9962EE050-4) to obtain polypeptide chain V_SIRPαD1-VL_MSLN-CL (SEQ ID NO. 13). The synthesized genes were cloned into vectors, respectively, to construct an HY202-1 expression vector.

Anti-human MSLN antibody heavy chain VH_MSLN-CH1-Fc (supra) with V_SIRPαD1Gene fusion, the gene sequence is delivered to Nanjing Kingsrei to synthesize (project number: C9)962EE050-6) to obtain polypeptide chain VH_MSLN-CH1-Fc-V_SIRPαD1(SEQ ID NO. 14); the antibody light chain adopts Amatuximab monoclonal antibody VL_MSLN-CL (SEQ ID NO.15), the gene sequence was submitted to Nanjing Kinsley synthesis (item No.: C9962EE 050-8). The synthesized genes were cloned into vectors, respectively, to construct an HY202-2 expression vector.

Will V_SIRPαD1The gene is fused with the heavy chain of an anti-human MSLN antibody, and the gene sequence is delivered to Nanjing Kinsley to be synthesized (the item number is C6587EH120-18) to obtain a polypeptide chain V_SIRPαD1-VH_MSLN-CH1-Fc (SEQ ID No. 16); the antibody light chain adopts Amatuximab monoclonal antibody VL_MSLNand-CL, the gene sequence is delivered to Nanjing Kinsley for synthesis (project No.: C9962EE 050-8). The synthesized genes are cloned into vectors respectively, thereby constituting an HY202-3 expression vector.

2. Protein expression and purification

Preparing CHO cell culture Medium (CD CHO Medium +8mM GlutaMAX; Gibco, 10743-; CHO-K1 cells, 1X 10, were prepared in logarithmic growth phase⁷cells and 30. mu.g plasmid were mixed in an electric cuvette and the electric program: 300V, pulse width 15ms, pulse interval 1s, pulse 2 times, carry on the electricity and change; the cell suspension after electroporation was transferred to T75 square flask, and T75 square flask was placed at 37 ℃ and 8% CO₂And culturing in a static incubator. Sampling and counting after 24-48 h; changing culture Medium (CHO CD04 Medium +25 μ M MSX; Quacell, A11004; Sigma, M5379) within 48h, and performing pressure screening until the cell survival rate is recovered to more than 90%; taking 2 mu L of supernatant, and detecting the protein expression condition by using a protein immunoblotting method (DOT blot).

3. Protein expression assay

And detecting protein expression by using DOT blot. The method comprises the following specific steps: selecting a nitrocellulose membrane (BBI Life Sciences, F619511-0005) with a proper size according to the workload, and marking the corresponding position and direction of each hole; taking cell supernatant stock solution, spotting 2 μ L of 96-well membrane per well, carrying standard substance (0.01mg/mL, 0.1mg/mL, 1mg/mL, 10mg/mL), and air drying; immersing the membrane in a confining liquid (2.5g of skimmed milk powder and 50mL of PBS), and placing the membrane in a shaking table for 1h at the rotating speed of 60-90 rpm; adding a goat anti-human IgG-HRP antibody (Jakson immune Research, 109-; washing the membrane with PBS 3-5 times, removing the skimmed milk powder, and placing the membrane in a shaking table at the rotating speed of 60-90 rpm for 10min each time; 1mL of ECL chemiluminescence solution (Thermo, 34577) was used per 96-well plate-sized NC membrane; and (3) photographing by using a chemiluminescence imager, setting the exposure time to be automatic exposure, and carrying out gray level analysis on the picture by using image software. The results are shown in FIG. 2, both the monoclonal antibody and the double antibody products are expressed on the basis of the standard, and the yield is approximately 1 g/L.

4. Stable cell line selection

Electrically transforming the gene transfected cells for 24-48 h, namely diluting the cells by using a pressurized culture medium, and adding the diluted cells into a 96-well cell culture plate, wherein each well contains 10000 cells; the cell fusion degree reaches 60%, and the supernatant is taken for DOT detection; and (3) selecting high expression quantity, gradually carrying out amplification culture, detecting HPLC-titer, analyzing related quality parameters, and finally selecting 3-5 high expression clones. After screening, selecting cells in logarithmic growth phase, further screening by a limiting dilution method, and selecting the cell strain with the highest expression level for freezing and storing for later use.

5. Protein production and purification

Stably expressing cell lines (3X 10)⁵/mL) into a 2L shake flask containing 300mL serum-free medium, placing the shake flask in a shaker for culturing until the cell density grows to 2X 10⁶Culture supernatants were harvested per mL, purified using Protein A column, and the purified proteins were dialyzed and replaced in PBS (pH 7.0). SDS protein electrophoresis analysis is carried out to ensure that the purity of the protein is more than 98 percent.

6. Target binding Activity

Detection of target binding activity by the Elisa method:

the target binding activity of MSLN is analyzed, and the specific steps are as follows: dissolving human MSLN antigen into a coating buffer solution, and diluting to 0.5 mu g/mL; then added to a 96-well ELISA assay plate at 100. mu.L per well, and the ELISA plate was placed in a refrigerator at 4 ℃ overnight. During detection, the sample was washed 3 times with a washing solution (TBS, 0.05% Tween-20, pH 7.4); blocking with blocking solution (Wash, 2% BSA) first 1 at 37 deg.CWashing for 3 times by using a washing solution for 5 hours; then adding diluted experimental group antibody (setting Amatuximab monoclonal antibody as positive control, IgG1 negative control), incubating for 1h at 37 ℃, and washing for 3 times by washing liquid; then adding goat antibody (Jakson Immuno Research, 109-; washing for 6 times, adding a substrate of HRP, performing light-shielding color development reaction for 10-15 min, and adding a stop solution (1M H)₂SO₄) The reaction was stopped and the optical density was measured at a wavelength of 450 nm. The results are shown in table 1 and a in fig. 3, and the target binding activity of the diabody (HY 202-1: EC50 ═ 0.03794 nM; HY 202-2: EC50 ═ 0.02912; HY 202-3: EC50 ═ 0.01527) is similar to that of the mab product (EC50 ═ 0.01200).

TABLE 1 target binding Activity

Name (R)	HY202-1	HY202-2	HY202-3	Amatuximab monoclonal antibody
					EC50	0.03794	0.02912	0.01527	0.01200

Target for CD47 analysis by flow cytometry detectionThe binding activity comprises the following specific steps: get 10⁶CHO-K1-humanCD47 (Kinsery) cells suspended in 500. mu.L PBS, centrifuged at 1000rpm for 2min, the supernatant removed, and the cells resuspended in 100. mu.L (4 degrees) 0.2% BSA-PBS; adding the antibody, blowing, mixing uniformly, and incubating for 1h on ice in a dark place; adding 100 μ L (4 deg.C) of 0.2% BSA-PBS, and centrifuging at 1000rpm for 2 min; removing supernatant, washing with 200 μ L (4 deg.C) of 0.2% BSA-PBS for resuspension, centrifuging at 1000rpm for 2min, removing supernatant (washing repeatedly for 2 times), and adding 100 μ L (4 deg.C) of 0.2% BSA-PBS; adding 2.5. mu.L of Secondary Antibody Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody (Invitrogen, A-21445), blowing, mixing, and incubating for 1 hr on ice in the dark; adding 100 μ L (4 deg.C) of 0.2% BSA-PBS, and centrifuging at 1000rpm for 2 min; the supernatant was removed, washed with 200. mu.L (4 ℃) of 0.2% BSA-PBS and resuspended, centrifuged at 1000rpm for 2min and repeated three times. And (4) performing detection on a flow cytometer. The results are shown in table 2 and figure 3B, the double antibody product was able to bind human CD47 on cells in a concentration-dependent manner with similar binding capacity to sirpa-Fc fusion proteins.

TABLE 2 target binding Activity

Name (R)	HY202-1	HY202-2	HY202-3	SIRP alpha-Fc fusion protein
					EC50	0.1642	0.1247	0.06750	0.04051

7. Antibody dependent cytotoxicity

Will be 10 of logarithmic growth phase⁵NCIH226 cells (target cells) were plated in 96-well plates at 100. mu.L per well; then adding Amatuximab monoclonal antibody and MSLNmAb-SIRP alpha with different concentrations at 37 ℃ and 5% CO₂Incubating for 30 min; ADCC Fc γ RIII (158F) Jurkat cells (Effector cells, Ginkam Biotech, Inc. of Shanghai) were adjusted to a density of 15X 10⁵and/mL. Effector cells were mixed with target cells at 15: 1 part by weight of a mixture of 5% CO at 37 deg.C₂Incubation for 18 h; the detection was performed using a luciferase detection kit (G0483M 001, gimeran, japan), and the ADCC activity was calculated according to the following formula:

response (relative luminescence units) (treatment sample value-background value)/(no antibody control-background value);

curves were plotted using GraphPad: the ordinate is the optical signal value, and the abscissa is the logarithm value of the drug concentration; from this, EC50 values for the antibody effect were calculated. The results are shown in tables 3-4 and fig. 4, the double-antibody product in human squamous cell lung carcinoma NCIH226 and breast cancer OVCAR-3 can obviously activate ADCC function, and the effect is better than that of the single-antibody product.

TABLE 3 NCIH226 cell reporter gene assay

Name (R)	HY202-1	HY202-2	HY202-3	Amatuximab monoclonal antibody
					EC50	0.1641	0.1402	0.1106	0.2080

TABLE 4 OVCAR3 reporter Gene assay

Name (R)	HY202-1	HY202-2	HY202-3	Amatuximab monoclonal antibody
					EC50	0.1193	0.2024	0.08054	0.1515

8. Antibody-dependent cellular phagocytosis

Mouse macrophage (Raw264.7) is addedInto 96-well cell culture plates at 5X 10 per well⁵Cell, 37 ℃, 5% CO₂Incubating for 2 h; CFSE (2.25. mu.M) labeled NCIH226 was mixed with different concentrations of SIRPa-Fc fusion protein, MSLNmAb-SIRPa, at 37 ℃ with 5% CO₂Incubate for 30min, then transfer to culture plate containing Raw264.7 cells, at 37 deg.C, 5% CO₂Incubating for 3 h; the target cells were washed three times with PBS, and then analyzed for CFSE signal in raw264.7 cells using flow cytometry. The results are shown in figure 5, and the double-antibody product remarkably promotes the phagocytic activity of macrophages and is superior to or similar to SIRP alpha-Fc fusion protein.

9. In vivo antitumor drug effect test

The in vivo anti-tumor activity of MSLNmAb-sirpa was studied using the NCIH226 subcutaneous tumor model. 30 nude mice were injected subcutaneously with NCIH226 cells, 3X 10 cells per mouse⁶Individual cell, tumor volume 100mm³The groups are randomly divided, the first group is injected with PBS in the abdominal cavity, the second group is injected with HY202-1(30mg/kg), the third group is injected with HY202-2(30mg/kg), the fourth group is injected with HY202-3(30mg/kg), the fifth group is injected with SIRP alpha-Fc (15mg/kg) in the abdominal cavity, and the sixth group is injected with Amatuximab (25mg/kg) in the abdominal cavity. The administration was 2 times per week for 4 consecutive weeks. Mice were observed daily for condition and body weight measured. As shown in FIG. 6, the double antibodies HY202-1, HY202-2 and HY202-3 exhibited certain antitumor activity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Hebei Shenyu Biotechnology Ltd

<120> recombinant bifunctional fusion protein and application thereof

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<211> 361

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

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

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

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

100 105 110

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

115 120 125

Ala Thr Pro Gln His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser

165 170 175

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

180 185 190

Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg

195 200 205

Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser

210 215 220

Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys

225 230 235 240

His Pro Leu Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys Arg Thr

245 250 255

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

260 265 270

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

275 280 285

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

290 295 300

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

305 310 315 320

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

325 330 335

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

340 345 350

Thr Lys Ser Phe Asn Arg Gly Glu Cys

355 360

<210> 4

<211> 597

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

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

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

465 470 475 480

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

485 490 495

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

500 505 510

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

515 520 525

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

530 535 540

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

545 550 555 560

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

565 570 575

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

580 585 590

Ala Thr Pro Gln His

595

<210> 5

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr

85 90 95

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

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 6

<211> 597

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

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

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

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

100 105 110

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

115 120 125

Ala Thr Pro Gln His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr

195 200 205

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

210 215 220

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

225 230 235 240

Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr

245 250 255

Trp Gly Ser Gly Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly

260 265 270

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

275 280 285

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

290 295 300

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

305 310 315 320

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

325 330 335

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

340 345 350

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

355 360 365

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

370 375 380

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

385 390 395 400

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

405 410 415

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

545 550 555 560

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

565 570 575

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

580 585 590

Leu Ser Pro Gly Lys

595

<210> 7

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

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

50 55 60

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

65 70 75 80

Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr

85 90 95

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

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 8

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

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

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 9

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 10

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggtgcagc tgcagcagtc tggaccagag ctggagaagc ctggagcctc tgtgaagatc 60

tcctgtaagg cttctggcta ctccttcacc ggctatacaa tgaactgggt gaagcagagc 120

catggcaagt ctctggagtg gatcggcctg atcacccctt acaacggcgc ctccagctat 180

aatcagaagt ttaggggcaa ggctaccctg acagtggaca agtcttccag caccgcctat 240

atggacctgc tgagcctgac atctgaggat tccgccgtgt acttctgcgc taggggcgga 300

tatgacggaa ggggctttga ttactggggc tccggcaccc ctgtgacagt gtcttccgct 360

tccaccaagg gcccaagcgt gtttccactg gcccccagct ctaagagcac ctctggagga 420

acagccgctc tgggctgtct ggtgaaggat tacttcccag agcccgtgac agtgagctgg 480

aactctggcg ccctgacctc cggagtgcac acatttcccg ctgtgctgca gtccagcggc 540

ctgtatagcc tgtcttccgt ggtgaccgtg cctagctctt ccctgggcac ccagacatac 600

atctgcaacg tgaatcacaa gccctccaat acaaaggtgg acaagaaggt ggagcctaag 660

agctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggcggacca 720

tccgtgttcc tgtttccacc caagcccaag gacaccctga tgatctccag aacccctgag 780

gtgacatgcg tggtggtgga cgtgtcccac gaggatccag aggtgaagtt caactggtac 840

gtggatggcg tggaggtgca taatgccaag accaagccaa gagaggagca gtacaattct 900

acctatcgcg tggtgtccgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa taaggccctg cccgctccta tcgagaagac catctctaag 1020

gctaagggcc agcccaggga gccacaggtg tacaccctgc ctccaagccg ggacgagctg 1080

accaagaacc aggtgtctct gacatgtctg gtgaagggct tctacccatc tgacatcgcc 1140

gtggagtggg agtccaatgg ccagcccgag aacaattata agaccacacc ccctgtgctg 1200

gactccgatg gcagcttctt tctgtactcc aagctgaccg tggataagag ccgctggcag 1260

cagggcaacg tgttttcctg tagcgtgatg catgaggctc tgcacaatca ttacacacag 1320

aagtctctgt ccctgagccc cggcaag 1347

<210> 11

<211> 399

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaggaggagc tgcaggtcat ccagccagac aagtccgtga gcgtggctgc tggagagagc 60

gccatcctgc attgtaccgt gacatctctg atcccagtgg gaccaatcca gtggtttagg 120

ggagctggac ctgctcggga gctgatctac aaccagaagg agggccactt cccaagagtg 180

accacagtgt ctgagtccac caagcgcgag aatatggact ttagcatctc tatctccgct 240

atcaccccag ccgatgctgg cacatactat tgcgtgaagt tcagaaaggg ctcccccgat 300

accgagttta agagcggagc tggaacagag ctgtctgtga gggccaagcc ttctgctcca 360

gtggtgtccg gccccgccgc tcgggctacc cctcagcac 399

<210> 12

<211> 639

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gacatcgagc tgacccagag cccagctatc atgtccgcct ccccaggaga gaaggtgacc 60

atgacatgtt ccgcctcctc ctccgtgtcc tacatgcatt ggtatcagca gaagtctggc 120

acctccccca agagatggat ctacgataca agcaagctgg cttctggagt gcctggccgc 180

ttctccggaa gcggatctgg caactcctat agcctgacca tctccagcgt ggaggccgag 240

gacgatgcta catactattg ccagcagtgg tctaagcatc ctctgacctt tggctccggc 300

acaaaggtgg agatcaagag gaccgtggcc gctccatccg tgttcatctt tccccctagc 360

gacgagcagc tgaagtccgg cacagccagc gtggtgtgcc tgctgaacaa tttctacccc 420

cgggaggcca aggtgcagtg gaaggtggat aacgctctgc agagcggcaa ttctcaggag 480

tccgtgaccg agcaggacag caaggattct acatattccc tgtcttccac cctgacactg 540

tctaaggccg actacgagaa gcacaaggtg tatgcttgcg aggtgaccca tcagggcctg 600

agctctcctg tgacaaagtc ctttaatcgc ggcgagtgt 639

<210> 13

<211> 1083

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaggaggagc tgcaggtcat ccagccagac aagtccgtga gcgtggctgc tggagagagc 60

gccatcctgc attgtaccgt gacatctctg atcccagtgg gaccaatcca gtggtttagg 120

ggagctggac ctgctcggga gctgatctac aaccagaagg agggccactt cccaagagtg 180

accacagtgt ctgagtccac caagcgcgag aatatggact ttagcatctc tatctccgct 240

atcaccccag ccgatgctgg cacatactat tgcgtgaagt tcagaaaggg ctcccccgat 300

accgagttta agagcggagc tggaacagag ctgtctgtga gggccaagcc ttctgctcca 360

gtggtgtccg gccccgccgc tcgggctacc cctcagcacg gaggaggagg aagcggcgga 420

ggaggctctg gcggcggcgg ctccgacatc gagctgaccc agagcccagc tatcatgtcc 480

gcctccccag gagagaaggt gaccatgaca tgttccgcct cctcctccgt gtcctacatg 540

cattggtatc agcagaagtc tggcacctcc cccaagagat ggatctacga tacaagcaag 600

ctggcttctg gagtgcctgg ccgcttctcc ggaagcggat ctggcaactc ctatagcctg 660

accatctcca gcgtggaggc cgaggacgat gctacatact attgccagca gtggtctaag 720

catcctctga cctttggctc cggcacaaag gtggagatca agaggaccgt ggccgctcca 780

tccgtgttca tctttccccc tagcgacgag cagctgaagt ccggcacagc cagcgtggtg 840

tgcctgctga acaatttcta cccccgggag gccaaggtgc agtggaaggt ggataacgct 900

ctgcagagcg gcaattctca ggagtccgtg accgagcagg acagcaagga ttctacatat 960

tccctgtctt ccaccctgac actgtctaag gccgactacg agaagcacaa ggtgtatgct 1020

tgcgaggtga cccatcaggg cctgagctct cctgtgacaa agtcctttaa tcgcggcgag 1080

tgt 1083

<210> 14

<211> 1791

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggtgcagc tgcagcagtc tggacctgag ctggagaagc caggagcctc cgtgaagatc 60

agctgtaagg cttccggcta cagcttcacc ggctatacaa tgaactgggt gaagcagtct 120

catggcaagt ccctggagtg gatcggcctg atcaccccat acaacggcgc ctccagctat 180

aatcagaagt ttagaggcaa ggctaccctg acagtggaca agtcttccag caccgcctat 240

atggacctgc tgtctctgac atccgaggat agcgccgtgt acttctgcgc taggggcgga 300

tatgacggaa ggggctttga ttactggggc tctggaaccc ctgtgaccgt gtcctccgcc 360

tccaccaagg gaccatccgt gttcccactg gcccccagct ctaagagcac ctctggagga 420

acagccgctc tgggctgtct ggtgaaggat tacttcccag agcccgtgac agtgtcttgg 480

aactccggcg ccctgacctc tggagtgcac acatttcctg ctgtgctgca gtccagcggc 540

ctgtattccc tgtcttccgt ggtgaccgtg ccaagctctt ccctgggcac ccagacatac 600

atctgcaacg tgaatcacaa gccctctaat acaaaggtgg acaagaaggt ggagcctaag 660

tcctgtgata agacccatac atgcccccct tgtcctgctc cagagctgct gggcggacca 720

agcgtgttcc tgtttccacc caagcccaag gacaccctga tgatcagccg cacccctgag 780

gtgacatgcg tggtggtgga cgtgtctcac gaggatccag aggtgaagtt caactggtac 840

gtggatggcg tggaggtgca taatgccaag accaagccca gggaggagca gtacaattct 900

acctatcggg tggtgtccgt gctgacagtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgtccaa taaggccctg cccgctccta tcgagaagac catcagcaag 1020

gctaagggcc agcccaggga gccacaggtg tacaccctgc ctccatcccg ggacgagctg 1080

accaagaacc aggtgagcct gacatgtctg gtgaagggct tctacccatc tgacatcgcc 1140

gtggagtggg agtccaatgg ccagcccgag aacaattata agaccacacc ccctgtgctg 1200

gacagcgatg gctctttctt tctgtactcc aagctgaccg tggacaagag caggtggcag 1260

cagggcaacg tgttttcctg cagcgtgatg catgaggctc tgcacaatca ttatacacag 1320

aagtctctgt ccctgagccc cggcaaggga ggaggaggat ccggaggagg aggaagcggc 1380

ggcggcggct ctgaggagga gctgcaggtc atccagcctg ataagtccgt gtccgtggct 1440

gctggagaga gcgccatcct gcactgtacc gtgacatctc tgatccccgt gggccctatc 1500

cagtggttta gaggagctgg accagctcgc gagctgatct acaaccagaa ggagggccat 1560

ttccctagag tgaccacagt gagcgagtct accaagcgcg agaatatgga cttttccatc 1620

agcatctctg ccatcacccc agccgatgct ggcacatact attgcgtgaa gttccggaag 1680

ggctctcccg ataccgagtt taagtccggc gctggcacag agctgagcgt gagagccaag 1740

ccatccgctc ccgtggtgag cggccccgcc gctcgcgcca cccctcagca t 1791

<210> 15

<211> 639

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gacatcgagc tgacccagag cccagctatc atgtccgcct ccccaggaga gaaggtgacc 60

atgacatgtt ccgcctcctc ctccgtgtcc tacatgcatt ggtatcagca gaagagcggc 120

acctctccta agaggtggat ctacgataca tccaagctgg cctccggagt gccaggccgg 180

ttctctggat ccggaagcgg caactcttat tccctgacca tctccagcgt ggaggccgag 240

gacgatgcta catactattg ccagcagtgg agcaagcacc ccctgacctt tggctctggc 300

acaaaggtgg agatcaagag aaccgtggcc gctccttccg tgttcatctt tcccccttcc 360

gacgagcagc tgaagtctgg cacagcctcc gtggtgtgcc tgctgaacaa tttctaccca 420

cgcgaggcca aggtgcagtg gaaggtggat aacgctctgc agtccggcaa tagccaggag 480

tctgtgaccg agcaggactc caaggatagc acatattctc tgtcttccac cctgacactg 540

tccaaggccg actacgagaa gcacaaggtg tatgcttgcg aggtgaccca tcagggcctg 600

agctctcccg tgacaaagag ctttaatagg ggcgagtgt 639

<210> 16

<211> 1791

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gaggaggagc tgcaggtcat ccagccagac aagagcgtgt ctgtggccgc tggcgagtct 60

gctatcctgc attgtaccgt gacatccctg atcccagtgg gaccaatcca gtggtttagg 120

ggagctggac ctgctaggga gctgatctat aaccagaagg agggccactt cccaagagtg 180

accacagtgt ccgagagcac caagcgcgag aatatggact tttctatctc catcagcgcc 240

atcaccccag ccgatgctgg cacatactat tgcgtgaagt tcaggaaggg cagccccgat 300

accgagttta agtctggcgc tggcacagag ctgtccgtga gagccaagcc ttccgcccca 360

gtggtgtctg gccccgccgc tcgcgccacc cctcagcatg gaggaggagg atccggagga 420

ggaggcagcg gcggcggcgg ctctcaggtg cagctgcagc agagcggacc tgagctggag 480

aagccaggag cctccgtgaa gatcagctgt aaggcttccg gctacagctt caccggctat 540

acaatgaact gggtgaagca gtctcatggc aagtccctgg agtggatcgg cctgatcacc 600

ccttacaacg gcgcctccag ctataatcag aagtttcggg gcaaggctac cctgacagtg 660

gacaagtctt ccagcaccgc ctatatggac ctgctgtctc tgacatccga ggatagcgcc 720

gtgtacttct gcgctagagg aggatatgac ggaaggggct ttgattactg gggctctggc 780

acccctgtga cagtgtcttc cgcttccacc aagggaccaa gcgtgttccc actggctcct 840

agctctaagt ctacctccgg aggaacagcc gctctgggct gtctggtgaa ggattacttc 900

ccagagcccg tgacagtgtc ttggaactcc ggcgccctga cctctggagt gcatacattt 960

cctgctgtgc tgcagtccag cggcctgtat tccctgtctt ccgtggtgac cgtgccaagc 1020

tcttccctgg gcacccagac atacatctgc aacgtgaatc acaagccctc caatacaaag 1080

gtggacaaga aggtggagcc taagagctgt gataagaccc atacatgccc cccttgtcct 1140

gctccagagc tgctgggcgg accaagcgtg ttcctgtttc cacccaagcc caaggacacc 1200

ctgatgatct ccagaacccc cgaggtgaca tgcgtggtgg tggacgtgtc ccacgaggat 1260

cctgaggtga agtttaactg gtacgtggat ggcgtggagg tgcataatgc caagaccaag 1320

cccagggagg agcagtacaa ttctacctat cgggtggtgt ccgtgctgac agtgctgcac 1380

caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaataaggc cctgcccgct 1440

cctatcgaga agaccatctc taaggctaag ggccagccaa gggagcccca ggtgtacacc 1500

ctgcctccaa gccgggacga gctgaccaag aaccaggtgt ctctgacatg tctggtgaag 1560

ggcttctacc catccgacat cgccgtggag tgggagagca atggccagcc cgagaacaat 1620

tataagacca caccccctgt gctggacagc gatggctctt tctttctgta cagcaagctg 1680

accgtggata agtctcgctg gcagcagggc aacgtgttta gctgttctgt gatgcatgag 1740

gccctgcaca atcattacac acagaagtcc ctgagcctgt ctcctggcaa g 1791

Claims

1. A recombinant bifunctional fusion protein, wherein said fusion protein is formed by linking an anti-human MSLN antibody to a single domain structure that binds to human CD47 protein, said recombinant bifunctional fusion protein comprising a first binding domain and a second binding domain;

2. The fusion protein of claim 1, wherein the single domain structure that binds to human CD47 protein comprises an extra-membrane N-terminal V-like domain (D1) derived from human signal-regulating protein;

preferably, the amino acid sequence of the N-terminal V-like domain (D1) derived from the outside of the human signal regulatory protein membrane is shown as SEQ ID NO. 1;

more preferably, the single domain structure that binds to human CD47 protein further comprises amino acids that have at least 80%, 85%, 90%, 95%, 98% or 99% similarity to the sequence shown in SEQ ID No. 1.

3. The fusion protein of claim 1, wherein the fusion protein comprises a heavy chain comprising the polypeptide chain VH and a light chain_MSLN-CH1-Fc, the light chain comprising polypeptide chain V_SIRPαD1-(X)_n-VL_MSLN-CL; wherein VH_MSLNCH1 is the first constant domain of the heavy chain constant region of an anti-human MSLN antibody heavy chain; v_SIRPαD1An extra-membrane N-terminal V-like domain derived from human signal-regulating protein for paired binding to CD47 (D1); x is a linker, n is more than or equal to 0; VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

4. The fusion protein of claim 3, wherein the peptide chain VH_MSLNThe amino acid sequence of-CH 1-Fc is shown in SEQ ID NO. 2; the polypeptide chain V_SIRPαD1-(X)_n-VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 3.

5. The fusion protein of claim 1, wherein the fusion protein comprises a heavy chain comprising the polypeptide chain VH and a light chain_MSLN-CH1-Fc-(X)_n-V_SIRPαD1Said light chain comprising a polypeptide chain VL_MSLN-CL; wherein VH_MSLNA variable domain that is an anti-human MSLN antibody heavy chain; CH1 is the first constant domain of the heavy chain constant region; x is a linker, n is more than or equal to 0; v_SIRPαD1Human signal regulatory protein derived extramembranous N-terminal V-like domain for paired binding to CD47(D1)；VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

6. The fusion protein of claim 5, wherein the polypeptide chain VH_MSLN-CH1-Fc-(X)_n-V_SIRPαD1The amino acid sequence of (A) is shown in SEQ ID NO. 4; said polypeptide chain VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 5.

7. The fusion protein of claim 1, wherein the fusion protein comprises a heavy chain comprising polypeptide chain V and a light chain_SIRPαD1-(X)_n-VH_MSLN-CH1-Fc, the light chain comprising a polypeptide chain VL_MSLN-CL; wherein V_SIRPαD1An extra-membrane N-terminal V-like domain derived from human signal-regulating protein for paired binding to CD47 (D1); x is a linker, n is more than or equal to 0; VH_MSLNA variable domain that is an anti-human MSLN antibody heavy chain; CH1 is the first constant domain of the heavy chain constant region; VL_MSLNA variable domain that is an anti-human MSLN antibody light chain; CL is the anti-human MSLN antibody light chain constant domain.

8. The fusion protein of claim 7, wherein the peptide chain V is_SIRPαD1-(X)_n-VH_MSLNThe amino acid sequence of-CH 1-Fc is shown as SEQ ID NO.6, and the polypeptide chain VL_MSLNThe amino acid sequence of-CL is shown in SEQ ID NO. 7.

9. The fusion protein of any one of claims 3 to 8, wherein the domain source of the Fc comprises a wild-type or mutant; linker X is a polypeptide chain consisting of a plurality of identical or different amino acids.

10. A medicament for treating MSLN-positive tumors, wherein the active ingredient of the medicament comprises the fusion protein according to any one of claims 1 to 9.