WO2018166468A1 - Igg-like long-acting immune fusion protein and use thereof - Google Patents

Abstract

Disclosed are an IgG-like long-acting immune fusion protein and the use thereof. The IgG-like long-acting immune fusion protein consists of an effector molecule and a constant region of an IgG antibody, and the effector molecule is linked to the constant region of the IgG antibody by means of a linker peptide; the effector molecule is a protein capable of exerting physiological functions in the body; and the constant region of the IgG antibody is a structure of the IgG antibody with two heavy chain variable regions and two light chain variable regions removed.

Description

IgG-like long-acting immunological fusion protein and application thereof Technical field

The invention relates to the field of biotechnology, and relates to an IgG-like long-acting immunological fusion protein and application thereof.

Background technique

Recombinant protein drugs are a very important class of biotechnological drugs and are generally administered intravenously and subcutaneously in the clinic. Intravenous and subcutaneous injections are often accompanied by protein degradation, resulting in reduced activity and low bioavailability; those with smaller molecular weight (less than 60kD) can even be filtered into the urine through the glomerulus, thus achieving the required blood concentration and treatment. The effect needs repeated administration, and some need to be injected 2-3 times a day, which not only causes inconvenience to the patient, reduces patient compliance, and thus affects the curative effect. Therefore, it is clinically necessary to develop long-acting recombinant protein drugs. The current methods for prolonging the half-life of protein drugs are:

Constructing mutants by constructing mutants to extend the half-life of the protein, commonly used methods are: 1. Increase the degree of glycosylation of protein drugs, so as to form a protective effect on the surface of protein drugs, hinder the degradation of protein drugs by proteases, and increase protein stability. At the same time, the molecular weight of the protein drug is increased, and glomerular filtration is reduced. Drugs that have been successfully developed and marketed include recombinant human EPO mutants (Aranesp from Amgen) and recombinant human insulin mutants (Lantus from Aventis). Aranesp uses site-directed mutagenesis to increase the number of N-linked oligosaccharides from 3 to 5, increasing the molecular weight from 30 kDa to 50 kDa, and extending the half-life from 4-13 h to an average of 49 h. 2. The time to release the free drug is prolonged by forming a sustained release microprecipitate. Lantus is a mutant of recombinant human insulin expressed from E. coli K12 strain, which mutates Asp to Gly at the 21st position of the human insulin A chain; and adds two Arg at the 30th position of the B-chain carbon end. The isoelectric point PI of insulin changed from the original pH 4.0 to pH 6.7. This mutation makes it a completely clear solution in an acidic environment. Once injected into the subcutaneous tissue (pH is increased), it becomes an insoluble microprecipitate due to the isoelectric point characteristics, and is released sustainably, after subcutaneous injection with recombinant human insulin. The concentration in vivo was relatively constant for more than 24 hours compared to the duration of -8h.

PEGylation modified PEGylation, ie polyethylene (PEG) covalently modified protein. PEGylated protein drugs mainly reduce the molecular weight of proteins, reduce the filtration of drugs from the renal tubules, and PEG can act as a barrier to block the antigenic determinants on the surface of protein molecules, reduce immunogenicity, reduce the clearance rate in vivo, and the barrier function of PEG can also protect proteins. It is not easily hydrolyzed by proteases. These characteristics of PEGylation are beneficial to prolong the half-life of protein drugs. Currently available PEGylated protein drugs such as PEGylated asparaginase (Enzon's Oncaspar), PEGylated IFNα2b (Schering's PEGIntron), PEGylated IFNα2a (Roche's Pegasys) and PEGylated G - CSF (Neulasta of Amgen).

Serum albumin fusion human serum, albumin (HSA) is a single-chain aglycosylated globular protein with 585 amino acid residues, molecular weight 65kDa, most therapeutic proteins and peptides generally have a short half-life. HSA has a serum half-life of up to 19 days. The results of clinical trials showed that the average half-life of Albuferonα (HSA/IFNα fusion protein) was 148 hours, which was longer than the average half-life of Pegasys (80-140 hours) and the average half-life of PEGIntron 40 hours (22-60 hours). Albutropin is a protein expressed by fusion of the HSA gene and the recombinant human growth hormone (rHGH) gene. In monkeys, subcutaneous injection of albutropin (0.3 mg/kg) and HGH (0.3 mg/kg) resulted in a 6-fold increase in half-life and a 8-fold slower clearance.

A fusion protein prepared by human Fc. The fusion protein prepared by albumin fusion protein and Fc mainly enhances the biological half-life of the drug through two mechanisms, one is to increase the molecular weight of the drug, so that it is not easily filtered by the renal tubule; the other is through the neonatal Fc Binding of the neonatal Fc receptor (FcRn) protects the target protein from clearance in the blood (Rath T, Baker K, Dumont JA, et al. Crit Rev Biotechnol. 2015 Jun; 35(2): 235-54). The FcRn receptor, encoded by the fcgrt gene, also known as the Brambell receptor, is an MHC class I-like receptor molecule that forms a heterodimer with a non-covalent bond with β ₂ m microglobulin. The binding of the FcRn receptor to albumin and immunoglobulin is strictly pH dependent, and FcRn does not bind to albumin and immunoglobulin under physiological conditions (pH 7.4). However, when albumin and immunoglobulin are endocytosed into the cells through the pinocytosis pathway, in acidic lysosomes (pH 6.0-6.5), the affinity of FcRn with albumin and immunoglobulin is increased to form a complex, FcRn protection. Albumin and immunoglobulin are not degraded and release albumin and immunoglobulin out of the cell through the membranous circulation. The binding of albumin and immunoglobulin to FcRn protects them from being metabolized, extending their biological half-life. Currently, listed albumin and immunoglobulin products include abatacept, aflibercept, belatacept, conbercept (Sichuan Kanghong), dulaglutide, etanercept and albuterrepononacog alfa, among which etanercept and aflibercept are blockbuster drugs, and global sales of etanercept in 2016 reached $8.874 billion, afl ibercept reaches $5 billion, and conbercept (Sichuan Kanghong) is expected to exceed 500 million yuan in sales in China. However, these Fc immuno-fusion proteins have a general disadvantage in that the biological half-life is shorter.

Invention disclosure

In order to effectively solve the shortcomings of the existing method for prolonging the biological half-life of the protein drug, the present invention designs an IgG-like immunological fusion protein, which can effectively prolong the biological half-life of the protein drug (effector molecule), which is far better than the similar Fc immunofusion protein.

The IgG-like immunological fusion protein provided by the present invention specifically comprises an effector molecule and an IgG antibody constant region, and the effector molecule is linked to the IgG antibody constant region through a linker peptide; the effector molecule is capable of exerting physiological functions in vivo The IgG antibody constant region is a structure in which an IgG antibody removes two heavy chain variable regions and two light chain variable regions.

That is, the IgG-like immunological fusion protein, like immunoglobulin, is a tetrapeptide chain structure composed of two light chains and two heavy chains, and the disulfide is between the heavy chains and between the heavy and light chains. The key is connected, and its conformation is similar to the English uppercase letter Y shape.

The light chain type may be of the kappa and lambda type, including the constant region (C _L ) of the light chain.

Heavy chain class may be an IgG1, IgG2 and IgG4, the heavy chain comprises a constant region (C _H 1, C _H 2 and C _H 3).

Wherein, the amino acid sequence of the linker peptide may be selected from any one of the following: AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (sequence) 6); RADAAPTVS (sequence 7); RADAAAAGGPGS (sequence 8); RADAAAA (sequence 9); SAKTTPKLEEGEFSEARV (sequence 10); ADAAP (sequence 11); DAAPTVSIFPP (sequence 12); TVAAP (sequence 13); TVAAPSVFIFPP (sequence 14) ;QPKAAP (sequence 15); QPKAAPSVTLFPP (sequence 16); AKTTPP (sequence 17); AKTTPPSVTPLAP (sequence 18); AKTTAP (sequence 19); AKTTAPSVYPLAP (sequence 20); ASTKGP (sequence 21); ASTKGPSVFPLAP (sequence 22); GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26).

The effector molecule is linked to the IgG antibody constant region by the linker peptide in any of the following eight ways:

Mode 1: The effector molecule is respectively linked to the two heavy chains of the constant region of the IgG antibody and the N-terminus of the two light chains by the linker peptide; the molecular formula is named: (EL) ₂ (EH) ₂ , E is Effector molecule. E- represents an N-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 2: The effector molecule is linked to the C-terminus of the two heavy chains of the IgG antibody constant region through the linker peptide, and no light chain is attached; the molecular formula is named: L ₂ (HE) ₂ , and E is an effector molecule. -E represents a C-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 3: The effector molecule is linked to the N-terminus of two light chains of the constant region of the IgG antibody through the linker peptide, and no heavy chain is attached; the molecular formula is named: (EL) ₂ H ₂ , and E is an effector molecule. E- represents an N-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 4: The effector molecule is linked to the C-terminus of two light chains of the IgG antibody constant region through the linker peptide, and no heavy chain is attached; the molecular formula is named: (LE) ₂ H ₂ , and E is an effector molecule. -E represents a C-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 5: The effector molecule is linked to the N-terminus of two heavy chains of the IgG antibody constant region through the linker peptide, and no light chain is attached; the molecular formula is named: L ₂ (EH) ₂ , and E is an effector molecule. E- represents an N-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 6: The effector molecule is linked to the N-terminus of the two heavy chains of the IgG antibody constant region and the C-terminus of the two light chains by the linker peptide, respectively. The molecular formula is named: (LE) ₂ (EH) ₂ , E is an effector molecule, -E represents a C-terminal linkage, E- represents an N-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 7: The effector molecule is respectively linked to the two heavy chains of the IgG antibody constant region and the C-terminus of the two light chains by the linker peptide; the molecular formula is named: (LE) ₂ (HE) ₂ , E is An effector molecule, -E represents a C-terminal linkage, L is a light chain, and H is a heavy chain.

Mode 8: The effector molecule is respectively linked to the N-terminus and the two heavy-chain C-termini of two light chains of the IgG antibody constant region by the linker peptide; the molecular formula is named: (EL) ₂ (HE) ₂ , E is an effector molecule, -E represents a C-terminal linkage, E- represents an N-terminal linkage, L is a light chain, and H is a heavy chain.

See Figure 1 for a schematic diagram of the structure of the IgG-like immunological fusion protein formed by the above eight ligation methods.

The targeting molecule acting on the effector molecule may be a targeting molecule for the treatment of diabetes, a targeting molecule for the treatment of autoimmune diseases, a targeting molecule for tumor therapy or a targeting molecule for endocrine therapy.

The target molecule for the treatment of diabetes may specifically be a glucagon-like peptide 1 receptor (GLP-1R) or a fibroblast growth factor receptor (FGFR) molecule. ;

The targeting molecule for treating the autoimmune disease may be specifically selected from any one of the following: Tumor necrosis factor (TNF-α), IL-1, IL-4, IL-5, IL-8, Molecules such as IL-13, IL-17, IL-23, CD80, CD86, CD2, CD110, CD257, and B lymphocyte stimulator (BLyS); mainly by inhibiting inflammatory mediators or by impeding lymphocyte surface receptors Somatic molecules inhibit the activation of lymphocytes.

The target molecule for tumor treatment may be specifically selected from any one of the following: vascular endothelial growth factor A (VEGF-A), Fas ligand (FasL), growth differentiation factor 2 (Growth) Differentiation molecules 2, GDF-2), Wnt, Bone morphogenetic protein 11, BMP-11, and activin A (Activin A); mainly by inhibiting tumor angiogenesis and promoting tumor cell apoptosis, Inhibition of tumor cell growth and metastasis to achieve the purpose of treating tumors.

The targeting molecule of the endocrine therapy may specifically be a molecule such as a growth hormone receptor (GHR) or a parathyroid hormone receptor (Parathyroid Hormone receptor). It exerts physiological effects through growth hormone and parathyroid hormone and its analogues for the treatment of diseases such as short stature, burns, and osteoporosis.

Correspondingly, the effector molecule acting on the diabetes treatment targeting molecule glucagon-like protein 1 receptor (GLP-1R) is GLP-1 or an analog thereof, and the amino acid sequence thereof is sequence 27 or sequence 28 in the sequence listing. Or the formula in Table 1; the effector molecule acting on the diabetes treatment targeting molecule fibroblast growth factor receptor (FGFR) is fibroblast growth factor 21 (FGF21), the amino acid sequence of which is specifically in the sequence listing Sequence 29. The details are shown in Table 1.

Table 1 Sequence of amino acid residues of effector molecules acting on diabetes treatment targeting molecules

Note: In the general formula of GLP-1 analogue, X _aa8 is Gly, Ser, Val or Ala; X _aa9 is Glu, Asp or Gln; X _aa18 is Ser or Lys; X _aa19 is Tyr or Asn; X _aa22 is Glu , Ala, Lys, Gly, Arg or Asp; X _aa23 is Gln or Lys; X _aa26 is Lys or Arg; X _aa29 is Ile or Val; X _aa34 is Lys or Arg; X _aa36 is Arg or Gly.

The effector molecule of the targeting molecule TNF-α acting on the treatment of autoimmune diseases is tumor necrosis factor receptor 1 (TNFR1) or tumor necrosis factor receptor 2 (TNFR2); the amino acid sequence of the TNFR1 is specifically a sequence listing Sequence 30 or sequence 31; the amino acid sequence of the TNFR2 is specifically sequence 32 or sequence 33 in the sequence listing; the effector molecules of the targeting molecules CD80 and CD86 acting on autoimmune diseases are cytotoxic T lymphocyte-associated The cytotoxic T lymphocyte-associated antigen-4 (CTLA-4); the amino acid sequence of the CTLA-4 is specifically the sequence 34 or the sequence 35 in the sequence listing; the target molecule CD2 acting on the treatment of autoimmune diseases The effector molecule is CD58 (LFA-3); the amino acid sequence of CD58 (LFA-3) is specifically sequence 36 in the sequence listing; targeting molecule B lymphocyte stimulating factor (BLyS) acting on autoimmune disease treatment The effector molecule is Transmembrane Activator And Caml Interactor (TACI); the amino acid sequence of the TACI is specifically sequence 37 in the sequence listing; targeting to the treatment of autoimmune diseases The effector molecule of CDCD257 is CD268; the amino acid sequence of CD268 is specifically sequence 38 in the sequence listing; the effector molecule of the targeting molecule CD110 acting on autoimmune disease treatment is immunoglobulin heavy chain constant region γ1 (immunoglobulin heavy constant gamma 1, IGHG1); the amino acid sequence of the IGHG1 is specifically the sequence 39 in the sequence listing. The details are shown in Table 2.

Table 2 Sequence of amino acid residues of effector molecules of targeting molecules for the treatment of autoimmune diseases

The effector molecule of VEGF-A, which acts on tumor therapy, is VEGFR-1 or VEGFR1-D2/VEGFR2-D3 (ie, functional region II (D2) representing VEGFR-1 and functional region III (D3) of VEGFR2 Or VEGFR1-D2/VEGFR2-D3-D4 (ie, functional region II (D2) of VEGFR-1 and functional region III (D3) of VEGFR2 and functional region IV (D4) of VEGFR2); The amino acid sequence is specifically the sequence 40 in the sequence listing; the amino acid sequence of the VEGFR1-D2/VEGFR2-D3 is specifically the sequence sequence 41 in the sequence listing; the amino acid sequence of the VEGFR1-D2/VEGFR2-D3-D4 is specifically in the sequence listing. Sequence sequence 42; the effector molecule of the target molecule FasL acting on tumor therapy is CD95; the amino acid sequence of the CD95 is specifically sequence 43 in the sequence listing; the targeting molecule growth differentiation factor 2 acting on tumor treatment (Growth differentiation) The effector molecule of factor 2, GDF-2) is Activin receptor-like kinase 1 (Activin Receptor-Like Kinase 1, ALK-1); the amino acid sequence of the ALK-1 is specifically the sequence 44 in the sequence listing; The effector molecule of Wnt, a targeting molecule that acts on tumor therapy, is Frizzled-8; the amino acid of Frizzled-8 The column is specifically sequence 45 in the sequence listing; the effector molecule of the target molecule bone morphogenetic protein 11 (BMP-11) acting on tumor therapy is activin A receptor type IIB (activin A receptor type IIB). , ACVR2B); the amino acid sequence of the ACVR2B is specifically sequence 46 or sequence 47 in the sequence listing; the effector molecule of the targeting molecule activin A acting on tumor therapy is activin A receptor type (activin A receptor type) 2A, ACVR2A); the amino acid sequence of the ACVR2A is specifically the sequence 48 in the sequence listing. The details are shown in Table 3.

Table 3 Sequence of amino acid residues of effector molecules acting on target molecules for tumor therapy

The effector molecule of the target molecule growth hormone receptor (GHR) acting on endocrine therapy is growth hormone; the amino acid sequence of the growth hormone is specifically sequence 49 in the sequence listing; targeting to endocrine therapy The effector molecule of the Parathyroid Hormone receptor is parathyroid hormone or an analogue thereof; the amino acid sequence of the parathyroid hormone or its analog is specifically the sequence 50 or sequence 51 in the sequence listing. . The details are shown in Table 4.

Table 4 Sequence of amino acid residues of effector molecules of targeting molecules acting on endocrine therapy

In an embodiment of the present invention, the IgG-like immunological fusion protein is specifically as follows:

(1) The amino acid sequence designated as BY23.2: the light chain of BY23.2 is as shown in SEQ ID NO: 58 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60 in the Sequence Listing;

(2) The amino acid sequence designated as BY23.1: the light chain of BY23.1 is as shown in the sequence 54 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 56 in the sequence listing;

(3) Named BY23.3: the amino acid sequence of the light chain of BY23.3 is as shown in the sequence 54 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 62 in the sequence listing;

(4) The amino acid sequence designated as BY23.4: the light chain of BY23.4 is as shown in the sequence 64 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 62 in the sequence listing;

(5) Named BY23.5: the amino acid sequence of the light chain of BY23.5 is as shown in SEQ ID NO: 58 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 56 in the Sequence Listing;

(6) designated as BY23.6: the amino acid sequence of the light chain of BY23.6 is as shown in the sequence 64 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 60 in the sequence listing;

(7) Named BY23.7: the amino acid sequence of the light chain of BY23.7 is as shown in the sequence 64 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 56 in the sequence listing;

(8) Named BY23.8: the amino acid sequence of the BY23.8 light chain is as shown in SEQ ID NO: 54 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60 in the Sequence Listing;

(9) designated as BY25: the amino acid sequence of the BY25 light chain is as shown in the sequence 70 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 72 in the sequence listing;

(10) designated as BY4.5: the amino acid sequence of the light chain of the BY4.5 is as shown in the sequence 74 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 76 in the sequence listing;

(11) Designated as BY3.4: The amino acid sequence of the light chain of BY3.4 is shown in SEQ ID NO: 78 in the Sequence Listing, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 80 in the Sequence Listing.

Further, the IgG-like immunological fusion protein can be obtained by a method comprising the steps of: inserting the coding gene of the light chain into a cleavage site XhoI of a glutamine synthetase high-efficiency expression vector having a double expression cassette; Between EcoRI, an intermediate vector is obtained; then the gene encoding the heavy chain is inserted between the restriction sites XbaI and SaII of the intermediate vector to obtain a recombinant expression vector; the protein expressed by the recombinant expression vector is The IgG-like immunological fusion protein. The glutamine synthetase high-efficiency expression vector having the double expression cassette is specifically referred to the Chinese patent of Application No. 201410441296.0.

More specifically, the coding gene of the light chain of BY23.2 is the DNA molecule shown by SEQ ID NO: 59 in the Sequence Listing; the coding gene of the heavy chain of BY23.2 is the DNA molecule shown by SEQ ID NO: 61 in the Sequence Listing. The coding gene of the light chain of BY23.1 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of the BY23.1 is the DNA molecule shown by the sequence 57 in the sequence listing. The coding gene of the light chain of BY23.3 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of BY23.3 is the DNA molecule shown by the sequence 63 in the sequence listing. The coding gene of the light chain of BY23.4 is the DNA molecule shown by the sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.4 is the DNA molecule shown by the sequence 63 in the sequence listing. The coding gene of the light chain of BY23.5 is the DNA molecule shown by the sequence 59 in the sequence listing; the coding gene of the heavy chain of BY23.5 is the DNA molecule shown by the sequence 57 in the sequence listing. The coding gene of the light chain of BY23.6 is the DNA molecule shown by the sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.6 is the DNA molecule shown by the sequence 61 in the sequence listing. The coding gene of the light chain of BY23.7 is the DNA molecule shown by the sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.7 is the DNA molecule shown by the sequence 57 in the sequence listing. The coding gene of the light chain of BY23.8 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of BY23.8 is the DNA molecule shown by the sequence 61 in the sequence listing. The coding gene of the light chain of BY25 is the DNA molecule shown by the sequence 71 in the sequence listing; the coding gene of the heavy chain of the BY25 is the DNA molecule shown by the sequence 73 in the sequence listing. The coding gene of the light chain of BY4.5 is the DNA molecule shown by the sequence 75 in the sequence listing; the coding gene of the heavy chain of the BY4.5 is the DNA molecule shown by the sequence 77 in the sequence listing. The coding gene of the light chain of BY3.4 is the DNA molecule shown by the sequence 79 in the sequence listing; the coding gene of the heavy chain of BY3.4 is the DNA molecule shown by the sequence 81 in the sequence listing.

The preparation method of the above IgG-like immunological fusion protein is also within the scope of protection of the present invention.

The use of the IgG-like immunological fusion protein in the treatment of diseases is also within the scope of the present invention.

The use of the IgG-like immunological fusion protein for the preparation of a medicament for the treatment of a disease is also within the scope of the present invention.

The invention also claims a method of treating a disease. The method can include the step of treating the disease with the IgG-like immunofusion protein.

The invention also claims a medicament for the treatment of a disease. The active ingredient of the drug is the IgG-like immunofusion protein.

Among them, the disease may be various diseases such as diabetes, autoimmune diseases, tumors or endocrine-related diseases.

DRAWINGS

Figure 1 is a schematic view showing the structure of an IgG-like immunological fusion protein formed by seven kinds of ligation methods.

Fig. 2 is a graph showing changes in blood glucose with time in the experimental group and the control group in Example 4. The ordinate is the blood glucose level in mmol/L.

The best way to implement the invention

The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.

Example 1. Construction of a high expression vector for glutamine synthetase

I. Cloning of a IgG-like immunological fusion protein encoding a diabetes-related gene and construction of its expression vector

1. Synthesis of Dulaglutide coding gene and construction of expression vector

Dulaglutide (Trulicity) is a US Food and Drug Administration (FDA) approved glucagon-like peptide-1 (GLP-1) receptor agonist and an immunological fusion protein used to help normalize blood glucose levels. Its structure is GLP-1-Fc, belonging to the existing typical Fc immunofusion protein.

The International Nonproprietary Name (INN) database and patents related to an amino acid residue sequence of Dulaglutide "HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGG GGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (Sequence 52)" Principal on Biological Engineering Co. Shanghai Generay Synthesis Dulaglutide genes encoding "CACGGAGAGGGCACATTCACTTCCGACGTGTCTAGTTACTTGGAGGAACAGGCAGCTAAAGAGTTTATCGCATGGCTCGTCAAGGGTGGGGGCGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGCGCTGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGgccgccGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCTAA (Sequence 53) ", after XhoI-EcoRI double Qieke Long enzyme to the dual expression cassette expression vector with glutamine synthetase (see in particular patent application number: 201410441296.0). The recombinant vector was constructed correctly by sequencing.

2. Synthesis and expression vector construction of GLP-1 analogue IgG-like fusion protein coding gene

The coding sequence was optimized based on the amino acid residue sequence of the effector molecule of the selected GLP-1 analog, and ligated to the coding genes of the light chain constant region and the heavy chain constant region of human IgG, respectively, to construct (EL) ₂ (EH) ₂ , L ₂ (HE) ₂ , (EL) ₂ H ₂ , (LE) ₂ H ₂ , L ₂ (EH) ₂ , (LE) ₂ (EH) ₂ and (LE) ₂ (HE) ₂ seven molecules A structured IgG-like fusion protein. The construction of eight molecular expression vectors is shown in Table 5 below. Table 6 lists the amino acid residues and coding sequences of several GLP-1 analog IgG-like fusion proteins.

Table 5 Construction of eight molecular expression vectors

Molecular formula Coding gene combination Protein name (EL) ₂ (EH) ₂ BY23.1L+BY23.1H BY23.1 L ₂ (HE) ₂ BY23.2L+BY23.2H BY23.2 (EL) ₂ H ₂ BY23.1L+BY23.3H BY23.3 (LE) ₂ H ₂ BY23.4L+BY23.3H BY23.4 L ₂ (EH) ₂ BY23.2L+BY23.1H BY23.5

(LE) ₂ (HE) ₂ BY23.4L+BY23.2H BY23.6 (LE) ₂ (EH) ₂ BY23.4L+BY23.1H BY23.7 (EL) ₂ (HE) ₂ BY23.1L+BY23.2H BY23.8

Table 6 Amino acid residues and coding sequences of several GLP-1 analog IgG-like fusion proteins:

The BY23.1L, BY23.2L and BY23.4L coding genes were ligated to the glutamine synthetase high-efficiency expression vector with double expression cassette by XhoI-EcoRI double digestion (see Patent Application No.: 201410441296.0); The combination of the BY23.1H, BY23.2H and BY23.3H coding genes was separately cloned into the glutamine synthetase high-efficiency expression vector of the double expression cassette encoding the light chain gene by XbaI-SaII (each recombinant expression vector was After sequencing and verifying correctly, the expressed IgG-like fusion proteins were named BY23.1, BY23.2, BY23.3, BY23.4, BY23.5, BY23.6, BY23.7 and BY23.8, respectively.

2. Cloning of IgG-like immunological fusion protein-coding genes and construction of expression vectors for autoimmune diseases

The inflammatory mediator TNF-α is a trimer that exerts a physiological function by binding to TNFR1 and TNFR2. TNFR1 and TNFR2 have high affinity to TNF-α, up to nM level. The functional regions where TNFR1 and TNFR2 bind to TNF-α are located in the four cysteine-rich regions at the N-terminus of the two receptors. It is designed as an IgG-like immunological fusion protein to bind free TNF-α in vivo for therapeutic purposes. The sequence of the amino acid residue of the effector molecule and the coding gene are shown in Table 7 below.

Table 7 Amino acid residues and coding sequences of IgG-like fusion proteins associated with autoimmune disease treatment

The BY19.3L coding gene was ligated to the glutamine synthetase high-efficiency expression vector with double expression cassette by XhoI-EcoRI double digestion (see Patent Application No.: 201410441296.0); the BY19.3H coding gene was cloned by XbaI-SaII, respectively. The glutamine synthetase high-efficiency expression vector (the recombinant expression vector was verified and verified by sequencing) to the double expression cassette of the BY19.3L-encoded light chain gene, and the expressed IgG-like fusion protein was named BY19.3, and the molecular formula L ₂ (HE) ₂ .

3. Cloning of IgG-like immunological fusion protein encoding gene for tumor therapy and construction of expression vector

VEGF-A has high affinity with its receptors VEGFR1 and VEGFR2. VEGFR belongs to the tyrosine kinase receptor superfamily, and there are 7 immunoglobulin-like domains in the extracellular segment. The 1-3 Ig-like regions are associated with VEGF binding. The extracellular domain 2 domain of VEGFR1 and the third functional domain encoding gene of VEGFR2 are ligated to form an IgG-like immunofusion protein, which can bind free VEGF-A and VEGF-B in vivo and inhibit tumor angiogenesis. In order to achieve the purpose of treating tumors. The sequence of the amino acid residue of the effector molecule and the coding gene are shown in Table 8 below.

Table 8 Amino acid residues and coding sequences of tumor-associated IgG-like fusion proteins

The BY25L-encoding gene was ligated to the glutamine synthetase high-efficiency expression vector with double expression cassette by XhoI-EcoRI double digestion (see Patent Application No.: 201410441296.0); the BY25H coding gene was cloned to BY25L by XbaI-SaII, respectively. The glutamine synthetase high-efficiency expression vector encoding the double expression cassette of the light chain gene (reported by the recombinant vector was verified), and the expressed IgG-like fusion protein was named BY25, and the molecular formula L ₂ (EH) ₂ .

4. Cloning of IgG-like immunological fusion protein encoding gene for endocrine diseases and construction of expression vector

Growth hormone plays a physiological role by binding to the growth hormone receptor, and lacks growth hormone in short stature. It needs to inject recombinant growth hormone once a day for the treatment of short stature, causing great pain to children with short stature, and reducing the number of injections during treatment. Even give up treatment. The use of long-acting growth hormone can improve the compliance and treatment of children. The sequence of the amino acid residue of the effector molecule and the coding gene are shown in Table 9 below.

Table 9 Amino acid residues and coding sequences of IgG-like fusion proteins associated with endocrine disease treatment

The BY4.5L and BY3.4L coding genes were ligated to the glutamine synthetase high-efficiency expression vector with double expression cassette by XhoI-EcoRI double digestion (see Patent Application No.: 201410441296.0); BY4 was separately obtained by XbaI-SaII. The 5H and BY3.4H coding genes were cloned into a double-expression cassette ligated to the BY4.5L and BY3.4L-encoded light chain genes, and the glutamine synthetase high-efficiency expression vector was verified by sequencing of the recombinant vector. The fusion proteins were named BY4.5 and BY3.4, and the molecular formulas were (EL) ₂ H ₂ and (EL) ₂ (EH) _{2 , respectively} .

Example 2. Screening, expression and purification of IgG-like immunofusion protein-expressing cells

I. Screening of IgG-like immunofusion protein-expressing cells

1. CHO-K1 cells (ATCC, Cat. No. CCL-61) were adapted and adherently grown in 10% D-FBS, 4 mM glutamine CD OptiCHO (Invitrogen product, Cat. No. 12681).

2. After 24 hours, the recombinant vector constructed in Example 1 was transfected into cells in a 10 cm culture dish according to the instructions of Lipofectin 2000 (Invitrogen Cat. No. 11668-019). Washing with serum-free antibiotic-free IMDM medium 2 Times, add IMμ 100 μl/well each time. The expression plasmid constructed in Example 1 was mixed with IMDM medium, and Lipofectin 2000 and IMDM medium were mixed. After standing for 5 minutes, mix the two. After standing at room temperature for 15 minutes, the Lipofectin 2000 CD and expression plasmid mixture was added to a 10 cm culture dish in which CHO-K1 cells were cultured.

After 3 and 5 hours, switch to complete medium (10% dialyzed serum, 4 mM glutamine CD OptiCHO).

4. Transfect the cells by trypsinization 24 hours after transfection, and plate 96-well culture plates at a cell density of 3-8×10 ³ /well, and use L-Methionine sulfoximine, MSX, Sigma products. , Item No.: M5379) Screening was performed on 25 μM CD OptiCHO medium.

5. After about 2 weeks, the cell clones grew. The protein content of the culture medium was determined by 50 μl sample double antibody sandwich ELISA method, and the clones with relatively high expression of target protein were screened.

2. Expression and purification of IgG-like immunological fusion protein

The high-expressing cells obtained in step 1 were cultured in serum-free CD OptiCHO, and the culture supernatant was collected at a cell viability of about 60%. The HiTrap MabSelect SuRe 1 ml column (GE Healthcare Life Sciences product, Cat. No: 11-0034-93) was equilibrated with a pH 7.4 PBS solution at 10 bed volumes at a flow rate of 0.5 ml/min; the culture supernatant was filtered with a 0.45 μm filter. Filtration was carried out at a flow rate of 0.5 ml/min. Wash 5-10 bed volumes with PBS solution of pH 7.4 at a flow rate of 0.5 ml/min; elute with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min, collect the elution peak, and store the target protein. In the elution peak.

Example 3, Biacore 3000 detects the affinity of an IgG-like immunofusion protein to its target

First, the experimental method

1. Fixed

GLP-1R, VEGF-A, VEGF-B and TNF-α (all products of Yishen Shenzhou) were diluted to a concentration of 10 μg/ml, and covalently immobilized on carboxymethyl glucoside via primary amine by amino coupling. Sugar-coated CM5 chip (General Electric product), fixed buffer 10 mM sodium acetate (pH 5.0), fixed amount of 1000 RU.

2, regeneration conditions

Regeneration solution: glycine buffer with a pH of 1.5-2.0; injection time: 30 s; flow rate: 30 μl/min.

3. Kinetic analysis

PBST (formulation: 0.005% by volume of Tween 20 in PBS) is Running buffer; using Kinetic Analysis Wizard mode; Dulaglutide, BY23.2, BY25 and BY19.3 are diluted to 600, 300, 150, respectively. Concentration gradients of 75, 37.5 and 18.75 nM; injection time: 2 min, dissociation time: 5 min, flow rate: 30 μl/min.

Second, the experimental results

Prototyping software: BIAevaluation 4.1 software.

The Biacore method detects the affinity results of Dulaglutide, BY23.2, BY25, and BY19 with the respective targets, as shown in Table 10.

Table 10 Affinity results for Dulaglutide, BY23.2, BY25, and BY19 with their respective targets

From the results of Dulaglutide and BY23.2 in Table 10, it can be seen that the affinity (KD) exhibited by the two is basically the same for the GLP-1R target. It can be seen that the IgG-like immunological fusion protein of the present invention retains a strong affinity compared to the conventional Fc immunological fusion protein.

Example 4, IgG-like immunological fusion protein activity assay

I. Detection of in vivo activity of IgG-like immunological fusion protein of GPL-1

1. Experimental method

(1) Type II diabetes model db/db mice (Changzhou Cavans Experimental Animal Co., Ltd.), measured basal blood glucose after fasting for 3 hours, and blood glucose concentration greater than 10 mmol/L into groups; with a few points 2 groups, each group 6 Only mice.

(2) The immunological fusion protein was subcutaneously injected in an amount of 0.8 mg/kg BY23.2 (γ ₄ , L ₂ (HE) ₂ ), and the control group was an equal volume buffer.

(3) Take tail blood of mice at 0 hours, 1 hour, 1 day, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days, and measure blood sugar (measured before blood sugar) The rats are fasted and can't help but water for 3 hours).

2, the experimental results

Compared with the control group, the experimental group BY23.2 (γ ₄ , L2 (HE) 2) subcutaneous injection within 1 hour of subcutaneous injection of blood glucose rapidly decreased to less than 10mmol / L, basically maintained blood glucose stability within 10 days, still significant after 10 days Hypoglycemic effect. Specifically as shown in Figure 2.

2. In vivo activity assay of growth hormone IgG-like immunological fusion protein

1. Experimental method

Commissioned by Suzhou Xishan Zhongke Drug Research and Development Co., Ltd. completed.

SD rats, male and female, 3 to 4 weeks old, pentobarbital sodium (concentration 15mg/mL) 45mg/kg intraperitoneal injection anesthesia (administered volume 3mL / kg), anesthetized and fixed in stereotactic pituitary resection instrument ( Stoelting, USA), the incisor plane is lower than the interaural line by 1mm. The depth and angle of the reference weight-depth and body weight are selected. The angle of the ear is adjusted to 19°. The 16G safety type is filled with the medicated pot. The needle core (1.7×50mm) is inclined upward by the needle tip of the right hollow ear stem. When the needle tip is just to the external auditory canal, the straight line distance between the pituitary part and the needle tip is measured with a compass, and the distance is used as the scale to start the needle to the sense of falling. While gently rotating the tip 180°, aspirate it to the pink pituitary tissue and aspirate it. The body weight and tail length of each animal (distance from the tail end to the anus) were recorded after surgery.

After the operation was completed, a single daily subcutaneous injection of penicillin (40 × 10 ⁴ IU / ml) 0.1ml / only 3 days after surgery, and freely drink 5% glucose water (in the water bottle); free drinking water and eating .

Three weeks after surgery, the body weight of each surviving animal was measured. The body weight change was less than ±10% of the pituitary rats before the operation. The body weight was randomly divided into the model control group. Normal saline) and BY4.5 (γ ₄ , (EL) ₂ H ₂ ) group (0.45 mg/kg in low dose group, 1.8 mg/kg in high dose group), human growth hormone international active standard (Chinese food and drug testing study) In the hospital, 4.5 IU (1.5 mg) / support group (low dose group 0.133 mg / kg, high dose group 0.533 mg / kg), 8 per group. The standard group was once a day for 6 consecutive days, and the BY4.5 group was injected twice a day for 6 times, once every 3 days.

On the 10th day, the animals were sacrificed and the hind legs were removed. The muscles and other tissues were removed. (If necessary, an autopsy can be performed after the experiment is completed, the sella region is cut, and the pituitary remains are examined visually, and the animals with pituitary remains are removed.) The top of the proximal end of the heart was cut along the sagittal plane and placed in 10% formalin. After washing for 10 minutes, the protein was deproteinized for 10 min in acetone, washed for 3 min, stained in 2% silver nitrate for 2 min, and glare was irradiated in water after washing. Brownish black, fixed with 10% sodium thiosulfate for 30s, stored in 80% ethanol for measurement. When measuring, cut about 1mm along the planing surface and measure the width of the humerus epiphyseal plate under the microscope (×100). Measure 10 values and take the average value).

2, the experimental results

The experimental results were sorted according to the format of the random design method for the quantitative reaction parallel line in the “Bioassay Statistical Method” (Appendix XIV of the Chinese Pharmacopoeia), and the results were randomly designed according to the quantitative parallel line measurement (2.2) method. Price calculation.

The final calculation results are shown in Table 11 below. The IgG-like immunological fusion protein still retains good biological activity in vivo.

Table 11 theoretical estimated titer of BY4.5 and actual calculation of potency

testing sample Theoretical estimate of titer Actual calculation of potency (IU/mg) BY4.5 1.24IU/mg 1.08

Example 5, GLP-1 analogue IgG-like immunological fusion protein cynomolgus monkey biological half-life determination

First, the experimental method

(1) Grouping: The experiment was divided into two groups of Dulaglutide and BY23.2 (γ ₄ , L ₂ (HE) ₂ ), and each group of 3 healthy cynomolgus monkeys.

(2) Animal administration: The test was administered in a single dose, single subcutaneous injection. According to the molecular weight, the amount (molar number) of administration is consistent, Dulaglutide is 0.52 mg/0.2 ml/kg, BY23.2 (γ ₄ , L ₂ (HE) ₂ ), and the dosage is 0.72 mg/ 0.2 ml/kg.

(3) Sample collection: before drug (0h), 0.5, 1, 6h, 1, 2, 3, 5, 7, 10, 14, 17, 21, 24, 28 days after administration. About 0.5 ml of whole blood was collected from the upper or lower extremity vein.

(4) Sample treatment: After taking blood, put into a 1.5ml centrifuge tube pre-added with 5μl of sodium heparin (6250IU/ml) (pre-blood, centrifuge tube containing heparin sodium stored at 2～8°C or ice bath), manual Mix at least 3 times, place in crushed ice, and centrifuge to separate plasma within 2 hours. The centrifugation conditions are: 2400g, 4°C, 10min, and the plasma is stored below -70°C to avoid repeated freezing and thawing.

(5) Determination of blood concentration: Abcam anti-GLP-1 antibody 4F3 was used as primary antibody (item number: ab23472), and horseradish peroxidase-labeled goat anti-human antibody (No.: ZB-2304) For the secondary antibody, the established ELISA method is used to determine the plasma drug concentration, and the test sample is used as a standard to prepare a standard curve.

(6) Data collection and statistical analysis: The content of each sample was calculated by using the enzyme-labeled instrument. The drug half-life (T1/2) is then calculated, which is the time required for the highest concentration of the drug to decrease by half in plasma.

Second, the experimental results

The T1/2 of Dulaglutide (Fc immuno-fusion protein) was 62.1±5.8h, and the T1/2 of BY23.2 (γ ₄ , L ₂ (HE) ₂ ) was 179±9.46h. BY23.2 was found in cynomolgus monkeys. The half-life is far more than Dulaglutide, which is more than three times that of Dulaglutide.

Based on the experimental results of the above various examples, it can be seen that the IgG-like immunological fusion protein provided by the invention can effectively prolong the protein drug under the premise of ensuring high affinity with the targeting molecule and having good in vivo activity ( The biological half-life of the effector molecule is much better than that of the similar Fc immunofusion protein. The IgG-like long-acting immunological fusion protein provided by the present invention can be used for the treatment of various diseases such as diabetes, tumor, autoimmune disease and endocrine.

Industrial application

The IgG-like immunological fusion protein provided by the invention can effectively prolong the biological half life of the protein drug (effector molecule) under the premise of ensuring high affinity with the targeting molecule and having good in vivo activity, far better than the biological half-life of the protein drug (effector molecule). Similar to the Fc immunological fusion protein. The IgG-like long-acting immunological fusion protein provided by the present invention can be used for the treatment of various diseases such as diabetes, tumor, autoimmune disease and endocrine.

Claims (18)

An IgG-like immunological fusion protein consisting of an effector molecule and an IgG antibody constant region, the effector molecule being linked to the IgG antibody constant region by a linker peptide; The effector molecule is a protein capable of exerting physiological functions in vivo; The IgG antibody constant region is a structure in which an IgG antibody removes two heavy chain variable regions and two light chain variable regions. The IgG-like immunological fusion protein according to claim 1, wherein the light chain of the IgG antibody is of a kappa type or a lambda type. The IgG-like immunological fusion protein according to claim 1 or 2, wherein the heavy chain of the IgG antibody is of the IgG1, IgG2 or IgG4. The IgG-like immunological fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the linker peptide is selected from any one of Sequence 1 to Sequence 26 in the Sequence Listing. The IgG-like immunological fusion protein according to any one of claims 1 to 4, wherein the effector molecule is linked to the IgG antibody constant region by the linker peptide in any of the following eight ways: Mode 1: The effector molecule is respectively linked to the two heavy chains of the constant region of the IgG antibody and the N-termini of the two light chains by the linker peptide; Mode 2: the effector molecule is linked to the C-terminus of two heavy chains of the IgG antibody constant region through the linker peptide, and no light chain is attached; Mode 3: the effector molecule is linked to the N-terminus of two light chains of the constant region of the IgG antibody through the linker peptide, and no heavy chain is attached; Mode 4: the effector molecule is linked to the C-terminus of the two light chains of the constant region of the IgG antibody through the linker peptide, and no heavy chain is attached; Mode 5: the effector molecule is respectively linked to the N-terminus of the two heavy chains of the IgG antibody constant region by the linker peptide, and no light chain is attached; Mode 6: the effector molecule is respectively linked to the N-terminus of the two heavy chains of the IgG antibody constant region and the C-terminus of the two light chains via the linker peptide; Mode 7: the effector molecule is respectively linked to the two heavy chains of the constant region of the IgG antibody and the C-terminus of the two light chains by the linker peptide; Mode 8: The effector molecule is linked to the N-terminus and the two heavy-chain C-termini of the two light chains of the IgG antibody constant region by the linker peptide, respectively. The IgG-like immunological fusion protein according to any one of claims 1 to 5, wherein the targeting molecule acting on the effector molecule is a targeting molecule for treating diabetes, a targeting molecule for treating autoimmune diseases, and a tumor. Targeted molecules for therapeutic targeting or endocrine therapy. The IgG-like immunological fusion protein according to claim 6, wherein the target molecule for treating diabetes is a glucagon-like peptide 1 receptor, a fibroblast growth factor receptor or other target molecule; The targeting molecule for treating the autoimmune disease is selected from any one of the following: tumor necrosis factor alpha, IL-1, IL-4, IL-5, IL-8, IL-13, IL-17, IL-23. , CD80, CD86, CD2, CD110, CD257, B lymphocyte stimulating factor, and other targeting molecules; The tumor-targeting molecule is selected from any one of the following: vascular endothelial growth factor A, Fas ligand, growth differentiation factor 2, Wnt, bone morphogenetic protein 11, activin A, and other targeting molecules; The targeting molecule for endocrine therapy is the growth hormone receptor or the parathyroid hormone receptor. The IgG-like immunological fusion protein according to claim 7, wherein the effector molecule acting on the diabetes treatment targeting molecule glucagon-like protein 1 receptor is a GLP-1 or GLP-1 analog; The effector molecule acting on the diabetes treatment targeting molecule fibroblast growth factor receptor is fibroblast growth factor 21; The effector molecule of the targeting molecule tumor necrosis factor alpha acting on the treatment of autoimmune diseases is tumor necrosis factor receptor 1 or tumor necrosis factor receptor 2; The effector molecule of the targeting molecules CD80 and CD86 acting on the treatment of autoimmune diseases is a cytotoxic T lymphocyte-associated antigen 4; The effector molecule of the targeting molecule CD2 acting on the treatment of autoimmune diseases is CD58; The effector molecule of the targeting molecule B lymphocyte stimulating factor acting on the treatment of autoimmune diseases is a transmembrane protein activator; The effector molecule of the targeting molecule CD257 acting on the treatment of autoimmune diseases is CD268; The effector molecule of the targeting molecule CD110 acting on the treatment of autoimmune diseases is an immunoglobulin heavy chain constant region γ1; The effector molecule of the targeting molecule vascular endothelial growth factor A acting on tumor therapy is VEGFR-1 or VEGFR1-D2/VEGFR2-D3 or VEGFR1-D2/VEGFR2-D3-D4; The effector molecule of the targeting molecule Fas ligand acting on tumor therapy is CD95; The effector molecule of the targeting molecule growth differentiation factor 2 acting on tumor therapy is activin A receptor-like kinase 1; The effector molecule of the targeting molecule Wnt acting on tumor therapy is Frizzled-8; The effector molecule of the targeting molecule bone morphogenetic protein 11 acting on tumor therapy is activin A receptor type IIB; The effector molecule of the targeting molecule activin A acting on tumor therapy is activin A receptor type IIA; The effector molecule of the targeting molecule growth hormone receptor acting on endocrine therapy is growth hormone; The effector molecule of the targeting molecule parathyroid hormone receptor acting on endocrine therapy is parathyroid hormone or an analogue thereof. The IgG-like immunological fusion protein according to claim 8, wherein the amino acid sequence of the GLP-1 is the sequence 27 or the sequence 28 in the sequence _listing ; and the GLP-1 analogue has the following formula: HX _aa8 - X _aa9 -GTFTSDVSX _aa18 -X _aa19 -LEX _aa22 -X _aa23 -AAX _aa26 -EFX _aa29 -AWLVX _aa34 -GX _aa36 ; wherein X _aa8 is Gly, Ser, Val or Ala; X _aa9 is Glu, Asp or Gln; _Aa18 is Ser or Lys; X _aa19 is Tyr or Asn; X _aa22 is Glu, Ala, Lys, Gly, Arg or Asp; X _aa23 is Gln or Lys; X _aa26 is Lys or Arg; X _aa29 is Ile or Val; _Aa34 is Lys or Arg; X _aa36 is Arg or Gly; The amino acid sequence of the fibroblast growth factor 21 is the sequence 29 in the sequence listing; The amino acid sequence of the tumor necrosis factor receptor 1 is sequence 30 or sequence 31 in the sequence listing; the amino acid sequence of the tumor necrosis factor receptor 2 is sequence 32 or sequence 33 in the sequence listing; The amino acid sequence of the cytotoxic T lymphocyte-associated antigen 4 is sequence 34 or sequence 35 in the sequence listing; The amino acid sequence of CD58 is sequence 36 in the sequence listing; The amino acid sequence of the transmembrane protein activator is sequence 37 in the sequence listing; The amino acid sequence of CD268 is sequence 38 in the sequence listing; The amino acid sequence of the immunoglobulin heavy chain constant region γ1 is the sequence 39 in the sequence listing; The amino acid sequence of the VEGFR-1 is the sequence 40 in the sequence listing; the amino acid sequence of the VEGFR1-D2/VEGFR2-D3 is the sequence 41 in the sequence listing; the amino acid sequence of the VEGFR1-D2/VEGFR2-D3-D4 is the sequence listing. Medium sequence 42; The amino acid sequence of CD95 is sequence 43 in the sequence listing; The amino acid sequence of the activin A receptor-like kinase 1 is the sequence 44 in the sequence listing; The amino acid sequence of Frizzled-8 is the sequence 45 in the sequence listing; The amino acid sequence of the activin A receptor type IIB is sequence 46 or sequence 47 in the sequence listing; The amino acid sequence of the activin A receptor type IIA is the sequence 48 in the sequence listing; The amino acid sequence of the growth hormone is sequence 49 in the sequence listing; The amino acid sequence of the parathyroid hormone or its analog is sequence 50 or sequence 51 in the sequence listing. The IgG-like immunological fusion protein according to any one of claims 1 to 9, wherein the IgG-like immunological fusion protein is any one of the following: (1) The amino acid sequence designated as BY23.2: the light chain of BY23.2 is as shown in SEQ ID NO: 58 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60 in the Sequence Listing; (2) The amino acid sequence designated as BY23.1: the light chain of BY23.1 is as shown in the sequence 54 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 56 in the sequence listing; (3) Named BY23.3: the amino acid sequence of the light chain of BY23.3 is as shown in the sequence 54 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 62 in the sequence listing; (4) The amino acid sequence designated as BY23.4: the light chain of BY23.4 is as shown in the sequence 64 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 62 in the sequence listing; (5) Named BY23.5: the amino acid sequence of the light chain of BY23.5 is as shown in SEQ ID NO: 58 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 56 in the Sequence Listing; (6) Named BY23.6: the amino acid sequence of the BY23.6 light chain is shown in SEQ ID NO: 64 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60 in the Sequence Listing; (7) Named BY23.7: the amino acid sequence of the light chain of BY23.7 is as shown in the sequence 64 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 56 in the sequence listing; (8) Named BY23.8: the amino acid sequence of the BY23.8 light chain is as shown in SEQ ID NO: 54 in the Sequence Listing, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60 in the Sequence Listing; (9) designated as BY25: the amino acid sequence of the BY25 light chain is as shown in the sequence 70 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 72 in the sequence listing; (10) designated as BY4.5: the amino acid sequence of the light chain of the BY4.5 is as shown in the sequence 74 in the sequence listing, and the amino acid sequence of the heavy chain is as shown in the sequence 76 in the sequence listing; (11) Designated as BY3.4: The amino acid sequence of the light chain of BY3.4 is shown in SEQ ID NO: 78 in the Sequence Listing, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 80 in the Sequence Listing. The IgG-like immunological fusion protein according to claim 10, wherein the IgG-like immunological fusion protein is obtained by a method comprising the steps of: inserting the coding gene of the light chain into a double expression cassette; An intermediate vector is obtained between the cleavage site XhoI and EcoRI of the glutamine synthetase high-efficiency expression vector; and the gene encoding the heavy chain is inserted between the restriction sites XbaI and SaII of the intermediate vector, A recombinant expression vector is obtained; the protein expressed by the recombinant expression vector is the IgG-like immunological fusion protein. The IgG-like immunological fusion protein according to claim 11, wherein the coding gene of the light chain of BY23.2 is a DNA molecule represented by the sequence 59 in the sequence listing; and the coding group of the heavy chain of the BY23.2 Because of the DNA molecule shown in SEQ ID NO: 61 in the sequence listing; The coding gene of the light chain of BY23.1 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of the BY23.1 is the DNA molecule shown by the sequence 57 in the sequence listing; The coding gene of the light chain of BY23.3 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of BY23.3 is the DNA molecule shown by the sequence 63 in the sequence listing; The coding gene of the light chain of BY23.4 is the DNA molecule of sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.4 is the DNA molecule of sequence 63 in the sequence listing; The coding gene of the light chain of BY23.5 is the DNA molecule shown by the sequence 59 in the sequence listing; the coding gene of the heavy chain of BY23.5 is the DNA molecule shown by the sequence 57 in the sequence listing; The coding gene of the light chain of BY23.6 is the DNA molecule shown by the sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.6 is the DNA molecule shown by the sequence 61 in the sequence listing; The coding gene of the light chain of BY23.7 is the DNA molecule shown by the sequence 65 in the sequence listing; the coding gene of the heavy chain of BY23.7 is the DNA molecule shown by the sequence 57 in the sequence listing; The coding gene of the light chain of BY23.8 is the DNA molecule shown by the sequence 55 in the sequence listing; the coding gene of the heavy chain of BY23.8 is the DNA molecule shown by the sequence 61 in the sequence listing; The coding gene of the light chain of BY25 is the DNA molecule shown by the sequence 71 in the sequence listing; the coding gene of the heavy chain of the BY25 is the DNA molecule shown by the sequence 73 in the sequence listing; The coding gene of the light chain of BY4.5 is the DNA molecule shown by the sequence 75 in the sequence listing; the coding gene of the heavy chain of the BY4.5 is the DNA molecule shown by the sequence 77 in the sequence listing; The coding gene of the light chain of BY3.4 is the DNA molecule shown by the sequence 79 in the sequence listing; the coding gene of the heavy chain of BY3.4 is the DNA molecule shown by the sequence 81 in the sequence listing. A method of producing the IgG-like immunological fusion protein of claim 11 or 12, comprising the steps of claim 11 or 12. Use of an IgG-like immunological fusion protein according to any one of claims 1 to 12 for the treatment of a disease. Use of an IgG-like immunological fusion protein according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of a disease. A method of treating a disease comprising the step of treating a disease with the IgG-like immunological fusion protein of any one of claims 1-12. A medicament for treating a disease, the active ingredient of which is the IgG-like immunological fusion protein of any one of claims 1-12. The method according to claim 14 or 15, or the method according to claim 16, or the drug according to claim 17, wherein the disease is diabetes, an autoimmune disease, a tumor or an endocrine-related disease.

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