A fusion protein ABD / Fc / IL-2, a coding gene thereof, a preparation method and application thereof

Abstract

The application discloses a fusion protein ABD / Fc / IL-2, a coding gene thereof, a preparation method and application, and belongs to the field of biopharmaceuticals. The fusion protein ABD / Fc / IL-2 provided comprises a human serum albumin binding domain ABD, a full-length human immunoglobulin Fc segment and IL-2 or a mutant thereof. The fusion protein can effectively stimulate the proliferation of immune cells in the body, enhance the immunity of the body, and has high biological activity and a long plasma circulation half-life, or has high biological activity, a long plasma circulation half-life and low toxic side effects. The fusion protein can be used for preparing medicines for treating various diseases such as malignant tumors, infectious diseases and low immunity of the body, and can play an important role in the fields of medicine and biopharmaceuticals.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmaceuticals, and particularly relates to a fusion protein (named fusion protein ABD / Fc / IL-2) comprising a human serum albumin binding domain ABD, a full-length human immunoglobulin Fc segment and IL-2 or a mutant thereof, a gene encoding the fusion protein, a preparation method and applications thereof, and particularly relates to the fusion protein ABD / Fc / IL-2 having high biological activity (protein specific activity higher than 1 x 10 8 IU / mg) and a long plasma circulation half-life (about 10 hours) or having high biological activity, a long plasma circulation half-life and low toxic side effects in the clinical application of preparing drugs for treating various diseases such as malignant tumors, infectious diseases and low immunity of the body. BACKGROUND

[0002] Interleukin-2 (IL-2) is a traditional immune-enhancing cytokine. Clinical practice has proved that IL-2 can effectively stimulate the proliferation of cytotoxic T cells, NK cells and other immune cells, enhance the immunity of the body, and play a more lasting tumor immune protection by inducing the immune memory function of the body, effectively delay tumor recurrence after surgery, inhibit tumor growth, and combined with immune checkpoint blockers and other drugs for treatment, synergistically play a tumor treatment role, and has been widely used in the comprehensive treatment of various malignant tumors such as renal cancer, melanoma, colon cancer, lung cancer and the treatment of infectious diseases such as AIDS and viral hepatitis.

[0003] However, the existing IL-2 products (such as recombinant IL-2) have the following major defects, which limit their clinical application: (1) The plasma half-life of IL-2 is very short (the in vivo circulation half-life is only 6.9 minutes), so in order to achieve an effective plasma concentration, frequent administration is required, resulting in poor compliance, and it is difficult to ensure stable IL-2 plasma concentration, and low-dose IL-2 will induce regulatory T cells, inhibit immune response, and promote tumor escape; (2) The recombinant IL-2 product produced by bacteria expression is obtained by refolding inclusion bodies, and the protein specific activity (unit activity) is low, and it is difficult to achieve the expected immune enhancement and regulation mediated by IL-2.

[0004] At present, there is no long-acting IL-2 product on the market at home and abroad. The products under research mainly use human serum albumin (HSA) or immunoglobulin Fc segment to recombinantly fuse with IL-2 to improve the plasma circulation half-life of IL-2.

[0005] Patent document CN1884520A (hereinafter referred to as document 1) discloses a human interleukin-2 and human serum albumin fusion protein (IL-2 / HSA), which is obtained by linking human IL-2 and HSA using recombinant technology and high-efficiency expression in Pichia pastoris. The main use is to modify IL-2 with human serum albumin with a longer plasma half-life (about 20 days) to achieve the long-acting effect of IL-2 (the peak time of blood concentration of IL-2 / HSA fusion protein subcutaneous injection is about 8 hours, and the plasma half-life is about 13 hours, for example, see Lei J et al., Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris, Protein expression and purification, 2012, 84(1): 154-160, hereinafter referred to as document 2). However, the document 1 uses bacteria (Pichia pastoris) to express and produce fusion protein IL-2 / HSA, which results in low specific activity (unit activity) of fusion protein IL-2 / HSA (about 1.147×10 6 IU / mg, see, for example, document 2), which may be difficult to achieve the expected immune enhancement and regulation mediated by IL-2, and when expressing fusion protein in Pichia pastoris, the product may have problems such as unevenness, excessive glycosylation and degradation, which may increase the immunogenicity of the fusion protein and affect the clinical effect of multiple drug administration, and is greatly limited in production and application. Although patent document CN104789594A (hereinafter referred to as document 3) has constructed a CHO cell strain stably and efficiently expressing human serum albumin and interleukin II fusion protein (IL-2 / HSA), which makes the expressed fusion protein closest to the natural protein, and few endogenous proteins are secreted, the product is secreted extracellularly, which is beneficial for purification, but the specific activity of the fusion protein obtained by expression and purification in the document 3 is still not high (the specific activity of IL-2-HSA fusion protein is 8.3×10 6 IU·mg -1 , and the specific activity of HSA-IL-2 fusion protein is 1.026×10 7 IU·mg -1 ), which may also be difficult to achieve the expected immune enhancement and regulation mediated by IL-2. On the other hand, the fusion protein IL-2 / HSA disclosed in the above-mentioned document 1, document 2 and document 3 has a large molecular weight (about 82 KDa), which is not conducive to tissue penetration in vivo and may limit its clinical efficacy and application value.

[0006] Patent document CN102174111A (hereinafter referred to as document 4) discloses a human interleukin 2-Fc fusion protein (IL-2 / Fc) which is obtained by linking human IL-2 with IgG Fc using recombinant technology and expressing in CHO cells. The fusion protein IL-2 / Fc mainly utilizes the reversible binding of Fc in the fusion protein IL-2 / Fc with FcRn receptors in tissues to increase the tissue stability of the fusion protein IL-2 / Fc, so as to prolong the action time of IL-2 (the actual plasma circulation half-life of the fusion protein IL-2 / Fc is about 2.5 hours according to the calculation of the attached figure 6 of the specification of the document 4), and the fusion protein provided in the document 4 also has ADCC and CDC effects due to the Fc segment in the fusion protein. However, the fusion protein IL-2 / Fc provided in the document 4, although compared with the existing recombinant IL-2 (recombinant human interleukin-2 (125Ser) / interleukin-2 for injection), can prolong the circulation half-life thereof in vivo, but the prolongation effect is still insufficient, and is greatly limited in clinical application. On the other hand, the fusion protein IL-2 / Fc disclosed in the document 4 only has the same affinity for binding with IL-2 receptors as the recombinant IL-2, so that the biological activity (mainly the specific activity of the protein) thereof is low, and it is also possible that the expected immune enhancement and regulation effects mediated by IL-2 cannot be achieved. SUMMARY

[0007] In view of one or more problems in the prior art, one aspect of the present application provides a fusion protein ABD / Fc / IL-2, which comprises a human serum albumin binding domain ABD (comprising an amino acid sequence as shown in SEQ ID NO: 1 in the sequence listing), a full-length human immunoglobulin Fc segment and IL-2 (comprising an amino acid sequence as shown in SEQ ID NO: 3 in the sequence listing) or a mutant thereof, and the fusion protein ABD / Fc / IL-2 has high biological activity (the specific activity of the protein is higher than 1x10 8 IU / mg); the full-length human immunoglobulin Fc segment is selected from human IgG1, IgG2, IgG3 and IgG4.

[0008] The full-length human immunoglobulin Fc segment described above comprises an amino acid sequence as shown in SEQ ID NO: 2 in the sequence listing.

[0009] The mutant of IL-2 is obtained by mutating any one or more of the 42nd, 45th, 72nd, 80th, 81st, 85th, 86th and 92nd sites in the amino acid sequence of native IL-2;

[0010] Preferably, F42, Y45, L72, L80, R81, L85, I86 and I92 in the amino acid sequence of native IL-2 are respectively mutated into A42, A45, A72, F80, D81, V85, V86 and F92.

[0011] Further preferably, the mutant of IL-2 is selected from any one of mutant IL-2v1, mutant IL-2v2 and mutant IL-2v3, wherein the mutant IL-2v1 is the amino acid sequence of F42, Y45 mutated to A42, A45, respectively, of the native IL-2; the mutant IL-2v2 is the amino acid sequence of L72 mutated to A72 of the mutant IL-2v1; the mutant IL-2v3 is the amino acid sequence of L80, R81, L85, I86, I92 mutated to F80, D81, V85, V86, F92, respectively, of the mutant IL-2v2.

[0012] Further preferably, the mutant of IL-2 comprises the amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in the Sequence Listing.

[0013] The fusion protein ABD / Fc / IL-2 described above further comprises a linker peptide or an analogue thereof for linking the human serum albumin binding domain ABD, the full-length human immunoglobulin Fc segment and IL-2 or its mutant into the fusion protein ABD / Fc / IL-2.

[0014] Preferably, the linker peptide or its analogue is used for linking the carboxy terminus of the human serum albumin binding domain ABD with the amino terminus of the full-length human immunoglobulin Fc segment, and for linking the carboxy terminus of the full-length human immunoglobulin Fc segment with the amino terminus of IL-2 or its mutant.

[0015] Optionally, the amino acid residue sequence of the linker peptide is as set forth in SEQ ID NO: 7 or SEQ ID NO: 8 in the Sequence Listing.

[0016] The amino acid residue sequence of the fusion protein ABD / Fc / IL-2 described above is one of the following amino acid residue sequences:

[0017] 1) the amino acid residue sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing;

[0018] 2) the amino acid sequence obtained by substitution, deletion or addition of one to ten amino acid residues of the amino acid residue sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing and having high biological activity (protein specific activity higher than 1 x 10 8 IU / mg).

[0019] Another aspect of the present application provides a gene coding for the fusion protein ABD / Fc / IL-2 (ABD / Fc / IL-2) described above, the nucleotide sequence of which is one of the following nucleotide sequences:

[0020] 1) the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing;

[0021] 2) a nucleotide sequence coding for SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing;

[0022] 3) a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or a nucleotide sequence coding for SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing, and the expressed protein has high biological activity (protein specific activity is higher than 1 x 10 8 IU / mg);

[0023] 4) a nucleotide sequence hybridizing with the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or a nucleotide sequence coding for SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing under high stringent conditions.

[0024] Another aspect of the present application also provides a recombinant expression vector pcDNA3.1-ABD / Fc / IL-2 comprising the gene coding for the fusion protein ABD / Fc / IL-2 (ABD / Fc / IL-2) described above for expressing the fusion protein ABD / Fc / IL-2 described above.

[0025] Another aspect of the present application also provides a transgenic cell line or host bacteria comprising the gene coding for the fusion protein ABD / Fc / IL-2 (ABD / Fc / IL-2) described above or the recombinant expression vector pcDNA3.1-ABD / Fc / IL-2 described above for expressing the fusion protein ABD / Fc / IL-2 described above.

[0026] Still another aspect of the present application provides a medicine for treating malignant tumors, infectious diseases and low immunity of the body, the medicine comprising the fusion protein ABD / Fc / IL-2 described above or the gene coding for the fusion protein ABD / Fc / IL-2 (ABD / Fc / IL-2) as an active ingredient.

[0027] Optionally, the medicine further comprises one or more of the following: a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption promoter, a surfactant.

[0028] In still another aspect, the present application provides a method for recombinantly expressing the fusion protein ABD / Fc / IL-2, comprising the following steps:

[0029] 1) constructing the transgenic cell line or host bacteria and culturing the transgenic cell line or host bacteria;

[0030] 2) isolating and purifying the protein from the culture medium or cells of the transgenic cell line or host bacteria to obtain the fusion protein ABD / Fc / IL-2;

[0031] Preferably, the method specifically comprises the following steps:

[0032] a) constructing the recombinant expression vector pcDNA3.1-ABD / Fc / IL-2 containing the above-mentioned encoding gene (ABD / Fc / IL-2);

[0033] b) transforming the recombinant expression vector pcDNA3.1-ABD / Fc / IL-2 into the transgenic cell line or host bacteria and culturing and expressing to obtain the fusion protein ABD / Fc / IL-2;

[0034] c) purifying the obtained fusion protein ABD / Fc / IL-2 by affinity chromatography or ion exchange chromatography.

[0035] In the present specification, the terms "fusion protein ABD / Fc / IL-2 (wild type)" and "fusion protein ABD / Fc / IL-2 (mutant)" are collectively referred to as "fusion protein ABD / Fc / IL-2".

[0036] Based on the above technical solution, the fusion protein ABD / Fc / IL-2 is composed of three parts of human serum albumin binding domain ABD, human immunoglobulin Fc segment (full length) and interleukin 2 (IL-2) fused together (which can be directly fused at the carboxyl end and amino acid of different parts or connected and fused through a connecting peptide or the like), wherein the ABD can be bound to human serum albumin, and then the modification of human serum albumin with a longer plasma half-life can make the fusion protein ABD / Fc / IL-2 have a longer action time; the human immunoglobulin Fc segment can increase the tissue stability of the fusion protein ABD / Fc / IL-2 through reversible binding with FcRn receptors in tissues, and can also make the fusion protein ABD / Fc / IL-2 have CDC and ADCC effects, thereby exerting immune effect functions; the IL-2 can effectively stimulate the proliferation of cytotoxic T cells, NK cells and other immune cells, thereby improving the basic immunity of the body.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] 1) The fusion protein ABD / Fc / IL-2 provided by this invention has high biological activity (protein specific activity is approximately 3.29 × 10⁻⁶). 8 The bioactivity (IU / mg) was significantly higher than that of the fusion protein IL-2 / HSA disclosed in References 1, 2, and 3 (the highest specific protein activity was 1.026 × 10⁻⁶). 7 IU·mg -1 The biological activity of recombinant IL-2 standards (protein specific activity approximately 8.5 × 10⁻⁶) and the biological activity of recombinant IL-2 standards were also examined. 6 IU / mg (see reference 3); it is also significantly higher than the bioactivity of the fusion protein IL-2 / Fc disclosed in reference 4 (it has the same affinity for binding to the IL-2 receptor as recombinant IL-2, reflected in its specific protein activity, which is approximately 8.5 × 10⁻⁶ IU / mg). 6 The fusion protein ABD / Fc / IL-2 provided by this invention is equivalent to (IU / mg) and therefore can more easily achieve the expected immune enhancement and regulation effects mediated by IL-2 compared to existing technologies.

[0039] 2) The fusion protein ABD / Fc / IL-2 provided by this invention can significantly prolong the in vivo action time of IL-2 (the peak plasma concentration after subcutaneous injection is about 8 hours, and significant biological activity can still be detected 48 hours after administration; its plasma circulating half-life is about 10 hours, which is comparable to the plasma circulating half-life of the fusion protein IL-2 / HSA disclosed in references 1, 2 and 3), and significantly longer than the plasma circulating half-life of the fusion protein IL-2 / Fc disclosed in reference 4 (the actual plasma circulating half-life calculated according to Figure 6 in reference 4 is about 2.5 hours, according to the appendix to this specification). Figure 5 According to the records, its plasma circulating half-life can also be calculated to be approximately 2.5 hours (consistent with that disclosed in Reference 4) and the existing in vivo plasma circulating half-life of recombinant IL-2 (6.9 minutes), thereby prolonging the in vivo plasma circulating half-life of IL-2, which is beneficial to ensuring a stable IL-2 plasma concentration.

[0040] 3) The fusion protein ABD / Fc / IL-2 provided by this invention has a full length of 438 amino acids, and its molecular weight is much smaller than that of the fusion protein IL-2 / HSA (with a full length of about 718 amino acids) provided in References 1, 2 and 3. Furthermore, the molecular weight of ABD in the fusion protein ABD / Fc / IL-2 provided by this invention is about 6 kDa, which is also much smaller than the molecular weight of HSA (about 66.5 kDa) in the fusion protein IL-2 / HSA provided in References 1, 2 and 3, which has a similar function. Therefore, it is more conducive to penetrating tissues in vivo, and it is also more conducive to expression and production, and more conducive to industrial production and clinical application.

[0041] 4) The fusion protein ABD / Fc / IL-2 provided by the present application can be produced by CHO cells, and the production process is simple. The structure of the fusion protein is closest to the spatial conformation of the natural protein, and the problems of non-uniformity, excessive glycosylation and degradation of the product produced by using Pichia pastoris and other bacteria can be avoided to the maximum extent, thereby avoiding the increase in immunogenicity of the product fusion protein.

[0042] 5) The present application also provides a low-toxicity fusion protein ABD / Fc / IL-2 (mutant) by introducing amino acid mutations at key sites of wild-type IL-2. The low-toxicity fusion protein not only has the advantages of high biological activity and long plasma circulation half-life of the fusion protein ABD / Fc / IL-2 (wild type), but also has the advantage of low toxicity compared with wild-type IL-2, the fusion protein IL-2 / HSA disclosed in documents 1, 2 and 3, and the fusion protein IL-2 / Fc disclosed in document 4. Therefore, the strong toxic side effects caused by high-dose IL-2, such as vascular leakage syndrome (VLS, leading to internal hydrops of human organs, such as pulmonary edema and liver cell damage), can be avoided, and the fusion protein is more suitable for use in the clinic to prepare drugs for treating various diseases such as malignant tumors, infectious diseases, and low immunity of the body.

[0043] In summary, the fusion protein ABD / Fc / IL-2 provided by the present application not only integrates the functions of serum albumin, the Fc fragment of immunoglobulin, and IL-2, and endows the fusion protein ABD / Fc / IL-2 with the ability to have a long plasma circulation half-life, CDC and ADCC effects, and IL-2 can effectively stimulate the proliferation of various immune cells such as cytotoxic T cells and NK cells, thereby improving the body's basic immunity, but also significantly improves the biological activity of the fusion protein ABD / Fc / IL-2 (the specific activity of the protein is higher than 1 x 10 8 IU / mg), and even reduces the toxic side effects of the fusion protein ABD / Fc / IL-2. Therefore, the fusion protein ABD / Fc / IL-2 provided by the present application is more suitable for clinical application, and its immunological ability and immunological effect are better than the IL-2-related products in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 SDS-PAGE (A) and Western blot (B) of the fusion protein ABD / Fc / IL-2 provided by an embodiment of the present application;

[0045] Figure 2 Four-parameter regression fitting curve of the recombinant human IL-2 standard;

[0046] Figure 3A four-parameter regression fitting curve of the fusion protein ABD / Fc / IL-2 provided by one embodiment of the present application;

[0047] Figure 4 Pull down / Western blot detection results of the fusion protein ABD / Fc / IL-2 provided by one embodiment of the present application;

[0048] Figure 5 Mouse pharmacokinetic curves of the fusion protein ABD / Fc / IL-2 and the fusion protein Fc / IL-2 provided by one embodiment of the present application, wherein A, B and C respectively represent the mouse pharmacokinetic curves of three different dose groups. DETAILED DESCRIPTION

[0049] The present application aims to provide a fusion protein ABD / Fc / IL-2 (wild type) with high biological activity and long plasma circulation half-life for clinical application of IL-2, further provide a fusion protein ABD / Fc / IL-2 (mutant) with high biological activity, long plasma circulation half-life and low toxic side effects, and provide an industrial production method of the fusion protein, so that IL-2 can better play a role in the treatment of various diseases such as malignant tumors, infectious diseases, and low body immunity. The present application is specifically realized by the following ways.

[0050] One object of the present application is to provide a fusion protein with high biological activity and long plasma circulation half-life, named fusion protein ABD / Fc / IL-2 (wild type), which comprises a human serum albumin binding domain (ABD), a full-length immunoglobulin Fc segment and human IL-2 (wild type), which has significantly higher biological activity and long plasma circulation half-life than the IL-2 related products (such as recombinant IL-2 or the fusion protein of IL-2 disclosed in the above-mentioned Document 1 to Document 4) existing in the prior art, and all retains the ability of IL-2 to stimulate the proliferation of immune cells such as T cells and NK cells, and can simultaneously exert the effects of CDC and ADCC.

[0051] Specifically, the fusion protein ABD / Fc / IL-2 (wild type) is obtained by connecting ABD at the amino terminal end (N-terminal end) of the immunoglobulin Fc segment and IL-2 at the carboxyl terminal end (C-terminal end) of the Fc segment through a connecting peptide. The fusion protein ABD / Fc / IL-2 (wild type) is a novel protein that can both stimulate immune cell proliferation and activate immune cells, and has high biological activity and a long plasma circulation half-life. Among them: ABD can be combined with serum albumin in the blood circulation to modify the fusion protein with macromolecules, prolong the in vivo plasma circulation half-life of the fusion protein, so as to achieve the purpose of prolonging the in vivo plasma circulation half-life without using serum albumin with a larger molecular weight to fuse with IL-2 as in the prior art (for example, the above-mentioned Document 1, Document 2 and Document 3), thereby significantly reducing the molecular weight of the fusion protein, facilitating expression and production, and more easily penetrating tissues in vivo; the immunoglobulin Fc segment can increase the tissue stability of the fusion protein by reversibly combining with FcRn in the tissue, and can also exert CDC and ADCC immune effects, thereby enhancing the immune efficacy of the fusion protein; the fusion protein also completely retains the ability of IL-2 to effectively stimulate the proliferation of cytotoxic T cells, NK cells and other immune cells, and improve the body's basic immunity. Therefore, the fusion protein ABD / Fc / IL-2 (wild type) provided by the present application integrates the effects of serum albumin, immunoglobulin Fc segment and IL-2, and also has the advantages of smaller molecular weight, which is more conducive to production and more suitable for clinical use, and also has the advantage of higher biological activity (protein specific activity) than the fusion proteins (IL-2 / HAS or IL-2 / Fc) and recombinant IL-2 in the prior art, so that the fusion protein ABD / Fc / IL-2 provided by the present application is more suitable for clinical production and application.

[0052] Preferably, the immunoglobulin Fc segment is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4, and the like, and the fusion protein ABD / Fc / IL-2 (wild type) can be directly fused from ABD, Fc segment, and IL-2, for example, the carboxyl terminal of ABD is directly fused to the amino terminal of Fc segment, the carboxyl terminal of Fc segment is directly fused to the amino terminal of IL-2, or the fusion can be through a linker peptide or the like, and the linker peptide can be selected from a variety of linker peptides, for example, the linker peptide having the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 in the sequence listing or the like can be selected. The ABD and Fc can also be replaced by analogs thereof, and the differences between the analogs and the ABD and Fc can be differences in the amino acid sequence, or can be differences in the form of modification that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. The induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, and can also be obtained by site-directed mutagenesis or other known molecular biology techniques. The analogs also include analogs having different L-amino acid residues (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids.

[0053] In particular, the human serum albumin binding domain ABD comprises the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, the full-length human immunoglobulin Fc segment comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and the IL-2 comprises the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. The fusion protein ABD / Fc / IL-2 (wild type) is one of the following amino acid residue sequences:

[0054] 1) the amino acid residue sequence shown in SEQ ID NO: 9 in the sequence listing;

[0055] 2) a protein obtained by substitution, deletion, or addition of amino acid residues to the amino acid residue sequence shown in SEQ ID NO: 9 in the sequence listing, and having serum albumin specificity, Fc function, and long-term stimulation / activation of immune cells.

[0056] SEQ ID NO: 9 consists of 438 amino acid residues, the amino terminal 1-56 (56 aa) amino acid residues are ABD, the amino terminal 57-59 (3 aa) amino acid residues are a linker peptide, the amino terminal 60-290 (231 aa) amino acid residues are an immunoglobulin Fc segment, the amino terminal 291-305 (15 aa) amino acid residues are a linker peptide, and the amino terminal 306-438 (133 aa) amino acid residues are IL-2.

[0057] Further, in one aspect, polypeptide fragments, derivatives and analogs of the fusion protein ABD / Fc / IL-2 (wild type) that have the same biological function or activity as the fusion protein ABD / Fc / IL-2 (wild type) are also encompassed by the present application. Polypeptide fragments are defined as: 1) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) which can or can not be encoded by the genetic code; 2) polypeptides having a substituent group at one or more amino acid residues; 3) polypeptides formed by fusing the mature polypeptide to another compound; 4) polypeptides formed by fusion of additional amino acid sequences to the polypeptide, such as sequences for purification of the polypeptide, or fusion proteins of antibody fragments or other antigen ligand sequences, or by fusion of nucleic acid sequences encoding another polypeptide (or portions thereof) to the nucleic acid sequences of the present application (or portions thereof) to produce a fusion polypeptide-encoding sequence, which is then expressed to produce the fusion polypeptide. Techniques for producing fusion polypeptides are well known in the art and include joining the polypeptide-encoding sequences such that they are in the same reading frame and the fusion polypeptide is expressed under the control of the same promoter and terminator.

[0058] 4) polypeptides formed by fusion of additional amino acid sequences to the polypeptide, such as sequences for purification of the polypeptide, or fusion proteins of antibody fragments or other antigen ligand sequences, or by fusion of nucleic acid sequences encoding another polypeptide (or portions thereof) to the nucleic acid sequences of the present application (or portions thereof) to produce a fusion polypeptide-encoding sequence, which is then expressed to produce the fusion polypeptide. Techniques for producing fusion polypeptides are well known in the art and include joining the polypeptide-encoding sequences such that they are in the same reading frame and the fusion polypeptide is expressed under the control of the same promoter and terminator.

[0059] Derivatives of the fusion protein ABD / Fc / IL-2 (wild type) include fusion proteins constructed using other IgG Fc segments or IL-2 mutants of the present application, and fusion proteins constructed using ABD / Fc and other cytokines that stimulate immune cell proliferation, such as IL-12, IL-15, etc.

[0060] Analog of the fusion protein ABD / Fc / IL-2 (wild type) can differ from the fusion protein ABD / Fc / IL-2 (wild type) in amino acid sequence, in modifications that do not affect the sequence, or both. The analogs include natural or induced genetic variants. Induced variants can be produced by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biology techniques. The analogs also include those having different natural or synthetic amino acids, such as D-amino acids. It is understood that the amino acid residue sequences of the fusion proteins of the present application are not limited to the representative sequences exemplified above.

[0061] Further, in another aspect, the fusion protein ABD / Fc / IL-2 (wild type) can also be a modified ABD / Fc / IL-2 polypeptide with improved anti-proteolytic properties or optimized solubility. Modifications (generally not changing the primary structure) include: 1) chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation; 2) glycosylation, such as polypeptides that have been modified by glycosylation during synthesis and processing or further processing steps, which can be accomplished by exposing the polypeptide to enzymes that glycosylate (such as mammalian glycosylation enzymes or deglycosylation enzymes); 3) sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0062] Another object of the present application is to provide a fusion protein with high biological activity, long plasma circulation half-life and low toxic side effects, named fusion protein ABD / Fc / IL-2 (mutant), which can be a derivative of the fusion protein ABD / Fc / IL-2 (wild type) provided by the present application, comprising a human serum albumin binding domain (ABD), a full-length human immunoglobulin Fc segment and a human IL-2 mutant, which not only has all the functions and advantages of the fusion protein ABD / Fc / IL-2 (wild type) provided by the present application, but also has the advantage of lower toxic side effects compared to the recombinant IL-2 products in the prior art and the fusion protein products disclosed in documents 1 to 4.

[0063] Clinical evidence shows that although the higher the dosage of IL-2 is used, the better the anti-tumor effect is, but the high dosage of IL-2 can cause strong side effects, such as vascular leakage syndrome (VLS), leading to internal hydrops of human organs, such as pulmonary edema and hepatocyte damage. Therefore, for IL-2 products, the bottleneck factor limiting the clinical application is still the large toxic side effects, and reducing the toxicity will be more conducive to the clinical application of IL-2 related products. Based on the method disclosed in patent document CN104231068A and the like, the inventors replace the natural IL-2 in the fusion protein ABD / Fc / IL-2 (wild type) provided in the present application with a low-toxicity IL-2 mutant obtained by amino acid mutation of a key site (binding point with the alpha and / or beta receptor of IL-2), thereby providing a fusion protein with high biological activity, long plasma circulation half-life and low toxic side effects.In particular, the low-toxicity IL-2 mutant can be a mutant obtained by mutation at any one or more of positions 42, 45, 72, 80, 81, 85, 86, 92 of the amino acid sequence of native IL-2; preferably, F42, Y45, L72, L80, R81, L85, I86, I92 of the amino acid sequence of native IL-2 are respectively mutated to A42, A45, A72, F80, D81, V85, V86, F92; further preferably, the low-toxicity IL-2 mutant includes the following three: (1) interleukin 2 mutant 1 (IL-2v1): the difference between the amino acid sequence thereof and the amino acid sequence of native IL-2 is that F42, Y45 of the amino acid sequence of native IL-2 are respectively mutated to A42, A45 in the amino acid sequence of IL-2v1, the amino acid residues of IL-2v1 are shown in SEQ ID NO: 4 in the sequence listing, and the amino acid sequence of the fusion protein ABD / Fc / IL-2 (mutant) is shown in SEQ ID NO: 11 in the sequence listing; (2) interleukin 2 mutant 2 (IL-2v2): the difference between the amino acid sequence thereof and the amino acid sequence of IL-2v1 is that L72 of the amino acid sequence of IL-2v1 is mutated to A72 in the amino acid sequence of IL-2v2, the amino acid residues of IL-2v2 are shown in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence of the fusion protein ABD / Fc / IL-2 (mutant) is shown in SEQ ID NO: 12 in the sequence listing; (3) interleukin 2 mutant 3 (IL-2v3): the difference between the amino acid sequence thereof and the amino acid sequence of IL-2v2 is that L80, R81, L85, I86, I92 of the amino acid sequence of IL-2v2 are respectively mutated to F80, D81, V85, V86, F92 in the amino acid sequence of IL-2v3, the amino acid residues of IL-2v3 are shown in SEQ ID NO: 6 in the sequence listing, and the amino acid sequence of the fusion protein ABD / Fc / IL-2 (mutant) is shown in SEQ ID NO: 13 in the sequence listing. By mutating the key positions of native IL-2, the affinity of the alpha receptor and IL-2 can be reduced or the affinity of IL-2 and the beta receptor can be enhanced, thereby affecting the ability to promote regulatory T cells, and further reducing and eliminating the toxic side effects of high-dose induced pulmonary edema.

[0064] The gene (named ABD / Fc / IL-2) encoding the above-mentioned fusion protein (fusion protein ABD / Fc / IL-2 (wild type) and fusion protein ABD / Fc / IL-2 (mutant)) also belongs to the present application, and the gene is one of the following nucleotide sequences:

[0065] 1) the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing;

[0066] 2) the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing;

[0067] 3) a nucleotide sequence having more than 90% homology with the nucleotide sequence represented by SEQ ID NO: 10 in the Sequence Listing or the nucleotide sequence encoding SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing and the expressed protein has high biological activity;

[0068] 4) a nucleotide sequence which hybridizes to the nucleotide sequence represented by SEQ ID NO: 10 in the Sequence Listing or the nucleotide sequence encoding SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing under high stringency conditions.

[0069] The high stringency conditions are washing the membrane with a solution containing 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS at 65°C after hybridization.

[0070] SEQ ID NO: 10 in the Sequence Listing consists of 1314 bases, the coding sequence of which is from the 1st to 1314th base from the 5' end, encoding a protein having the amino acid residue sequence represented by SEQ ID NO: 9 in the Sequence Listing, from the 1st to 168th base from the 5' end encoding an ABD, from the 169th to 177th base from the 5' end encoding a linker peptide, from the 178th to 870th base from the 5' end encoding a human immunoglobulin Fc segment, from the 871st to 915th base from the 5' end encoding a linker peptide, and from the 916th to 1314th base from the 5' end encoding an IL-2. The nucleotide sequence encoding the amino acid residue sequence represented by SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing can be easily determined from SEQ ID NO: 10 according to the residue difference between the amino acid sequence of the three and the amino acid sequence represented by SEQ ID NO: 9.

[0071] The polynucleotide encoding the fusion protein ABD / Fc / IL-2 of the present application (including the fusion protein ABD / Fc / IL-2 (wild type) and the fusion protein ABD / Fc / IL-2 (mutant)) can be in the form of DNA or RNA. The DNA form includes cDNA or artificially synthesized DNA, and can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0072] Further, variants of the polynucleotide encoding the fusion protein ABD / Fc / IL-2 of the present application, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the fusion protein ABD / Fc / IL-2, also belong to the present application. The variants of the polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants, and can include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which can result from natural or induced genetic variations and can result in changes to the function of the encoded polypeptide.

[0073] Expression vectors, transgenic cell lines and host organisms containing the gene (ABD / Fc / IL-2) of the present application also belong to the present application.

[0074] Another object of the present application is to provide a method for preparing the fusion protein ABD / Fc / IL-2 by recombinant expression, which comprises transforming or transducing a host cell with a recombinant expression vector containing the gene (ABD / Fc / IL-2) encoding the fusion protein ABD / Fc / IL-2, culturing the host cell, and isolating and purifying the protein from the culture medium or the cell to obtain the fusion protein ABD / Fc / IL-2.

[0075] In the method, the recombinant expression vector containing the gene (ABD / Fc / IL-2) encoding the fusion protein ABD / Fc / IL-2 is obtained by inserting the gene encoding the fusion protein ABD / Fc / IL-2 or a variant thereof into a recombinant expression vector. The starting vector for constructing the recombinant expression vector can be any of the well-known bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors that can express foreign genes. The starting vector includes, but is not limited to, expression vectors based on T7 promoters for expression in bacteria, vectors for expression in mammalian cells and vectors derived from baculovirus for expression in insect cells. In general, any plasmid and vector that can replicate and be stable in the host can be used. An important feature of the starting vector is that it usually contains a replication point, a promoter, a marker gene and a translation control element.

[0076] Specifically, the recombinant expression vector containing the coding gene of the fusion protein ABD / Fc / IL-2 can be constructed using pcDNA3.1 vector as the starting vector, and named pcDNA3.1 / ABD / Fc / IL-2. The recombinant expression vector pcDNA3.1 / ABD / Fc / IL-2 can be constructed by methods well known to those skilled in the art, such as in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombination technology (Sambrook, et al Molecular cloning, a Laboratory Manual. Cold spring harbor laboratory. New York, 1989). The DNA sequence of the coding gene of the fusion protein ABD / Fc / IL-2 can be effectively connected to a suitable promoter in the expression vector to direct the synthesis of mRNA. The promoter can be: lac or trp promoter of E. coli, phage promoter, retrovirus and other some known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes ribosome binding site for translation initiation and transcription terminator.

[0077] In addition, the recombinant expression vector pcDNA3.1 / ABD / Fc / IL-2 can also contain one or more selective marker genes to provide a phenotypic form for selecting transformed host cells, such as dihydrofolate reductase gene for eukaryotic cell culture, neomycin resistance gene and green fluorescent protein (GFP) gene or tetracycline or ampicillin resistance gene for E. coli, etc. When the coding gene of the fusion protein ABD / Fc / IL-2 of the present application is expressed in higher eukaryotic cells, in order to enhance transcription, an enhancer sequence can also be inserted into the recombinant expression vector pcDNA3.1 / ABD / Fc / IL-2. Enhancer is a cis-acting factor of DNA, usually 10-300 base pairs in length, which acts on the promoter to enhance gene transcription. Such as SV40 enhancer with a length of about 100-270 base pairs on the late side of the replication initiation point, polyoma enhancer on the late side of the replication initiation point or adenovirus enhancer, etc.

[0078] In this method, the transformed or transduced host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; higher eukaryotic cells, such as mammalian cells. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; eukaryotic cells such as yeast, plant cells; Drosophila S2 or Sf9 insect cells; CHO, COS, 293 cells or Bowes melanoma cells and other animal cells.

[0079] The recombinant expression vector pcDNA3.1 / ABD / Fc / IL-2 can be transformed into host cells by using conventional techniques known to those skilled in the art, the transformants can be cultured, the expression of the target protein (i.e. the fusion protein ABD / Fc / IL-2) can be induced, and the target protein can be isolated and purified.

[0080] The culture medium and culture conditions for the host cells containing the gene encoding the fusion protein ABD / Fc / IL-2 having high biological activity and long plasma circulation half-life or having high biological activity, long plasma circulation half-life and low toxic side effects can be the culture medium and culture conditions for the original host.

[0081] In order to stimulate / activate the high activity and long-acting (long in vivo circulation half-life) of the immune cells of the body or to have high activity, long-acting and low toxicity of the fusion protein ABD / Fc / IL-2 and the gene encoding the same, the present application also provides a medicine for treating various diseases such as malignant tumors, AIDS, and infectious diseases such as viral hepatitis. The active ingredient of the medicine comprises the fusion protein ABD / Fc / IL-2 or the gene encoding the same.

[0082] When the active ingredient of the medicine is the gene encoding the fusion protein ABD / Fc / IL-2, the gene encoding the fusion protein ABD / Fc / IL-2 can exist in various expression vectors such as prokaryotic and eukaryotic expression vectors.

[0083] If necessary, one or more pharmaceutically acceptable carriers can be added to the above-mentioned medicine. The carriers include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, and adsorption carriers, which are conventional in the pharmaceutical field.

[0084] The medicine of the present application can be prepared in various forms such as injection solutions or lyophilized powders. The medicines in the above-mentioned various forms can be prepared according to conventional methods in the pharmaceutical field.

[0085] In the following examples, the methods used are conventional unless otherwise specified, and the specific procedures can be found in "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0086] The percentage concentrations are mass / volume (W / V) percentage concentrations or volume / volume (V / V) percentage concentrations unless otherwise specified.

[0087] The DNA sequence synthesis and DNA sequence and amino acid sequence determination were completed by Nanjing Kingsriver Biotechnology Co., Ltd.

[0088] The obtaining approaches of various biomaterials described in the examples only provide an experimental obtaining approach to achieve the specific disclosed purposes, and should not be regarded as a limitation on the sources of the biomaterials of the present application. In fact, the sources of the biomaterials used are extensive, and any biomaterial that can be obtained without violating laws and moral ethics can be used according to the hints in the examples; in industrial implementation, various cells derived from rats, mice, pigs or humans and the like mammals are all ex vivo, and include being obtained from a cell bank or being commercially purchased, and also include being prepared according to the introduction in the existing literature, and being induced from various stem cells that can be commercially obtained by known methods.

[0089] The examples are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, and the examples will help to understand the present application, but the scope involved in the present application is not limited to the following examples.

[0090] Example 1: Expression and purification of fusion protein ABD / Fc / IL-2 (wild type) in CHO cells

[0091] 1.1 Construction of recombinant expression vector pcDNA3.1 / ABD / Fc / IL-2

[0092] ABD / Fc / IL-2 gene was optimized according to the preferred codons of CHO and synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and an expression vector pcDNA3.1 / ABD / Fc / IL-2 was constructed. Specifically, the ABD / Fc / IL-2 gene has the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing, which consists of 1314 bases, and the coding sequence is from the 1st to 1314th base at the 5' end, which encodes a protein having the amino acid residue sequence shown in SEQ ID NO: 9 in the sequence listing, wherein the ABD of the fusion protein ABD / Fc / IL-2 (wild type) is encoded by the 1st to 168th base at the 5' end, the linker peptide is encoded by the 169th to 177th base at the 5' end, the human immunoglobulin Fc segment is encoded by the 178th to 870th base at the 5' end, the linker peptide is encoded by the 871st to 915th base at the 5' end, and IL-2 is encoded by the 916th to 1314th base at the 5' end.

[0093] 1.2 Expression and purification of fusion protein ABD / Fc / IL-2 (wild type) in CHO cells

[0094] ExpiCHO-S TMOperation manual, the recombinant vector pcDNA3.1 / ABD / Fc / IL-2 non-pyrogenic plasmid constructed in step 1.1 above was transiently transfected into ExpiCHO-S cells TM , which were cultured at 37℃, 8% CO2, 120 rpm; the cells and supernatant were collected on the 5th day after transfection; the fusion protein ABD-Fc-IL-2 was purified by Protein A affinity chromatography, and identified by 12% SDS-PAGE electrophoresis and Western blot analysis.

[0095] The results are shown in Figure 1 , where Figure A represents the SDS-PAGE electrophoresis identification result of the purified fusion protein ABD-Fc-IL-2, and Figure B is the Western blot analysis result of Figure A; in Figures A and B, M represents Protein Maker; Lane 1 represents reducing electrophoresis; Lane 2 represents non-reducing electrophoresis. This example proves that the fusion protein ABD-Fc-IL-2 is obtained by expression and purification, and has high purity.

[0096] Example 2: Protein specific activity determination of fusion protein ABD-Fc-IL-2

[0097] This example determines the protein specific activity of the fusion protein ABD-Fc-IL-2 obtained by expression and purification in Example 1 by referring to the CTLL-2 / MTT cell proliferation colorimetric method in the Chinese Pharmacopoeia (see, for example, patent document CN102372780A), which specifically includes the following steps.

[0098] 1) The national standard for determination of biological activity of recombinant human IL-2 (purchased from China Institute for Food and Drug Control) 2000 IU / mL was diluted 10 times to 200 IU / mL according to the requirements of the instruction, and the fusion protein ABD-Fc-IL-2 with a concentration of 0.1 mg / mL was simultaneously diluted to 1000 times.

[0099] 2) In a 96-well cell culture plate, the recombinant IL-2 standard (200 IU / mL) and the fusion protein ABD-Fc-IL-2 diluted in step 1) were each subjected to 2-fold serial dilution, with a total of 8 dilution degrees for each, and 2 replicate wells for each dilution degree.

[0100] 3) 50 μL of CTLL-2 cell (5 x 10 5 / ml, ATCC TIB-214) suspension was added to each well, which was cultured at 37℃, 5% CO2 for 18-24 hours. Then, 20 μL of MTT solution was added to each well, which was cultured at 37℃, 5% CO2 for 4-6 hours.

[0101] 4) 150 μL of cell lysis solution was added to each well, which was incubated at 37℃ for 20 hours.

[0102] 5) The absorbance of each well was measured at 570 nm and the results were recorded.

[0103] 6) The results were processed by computer program or four-parameter regression calculation. The half-effective dilution fold of the test sample (i.e. fusion protein ABD-Fc-IL-2) was calculated, i.e. the dilution fold from the test sample solution to the dilution corresponding to the 50% maximum effect point of the standard sample, and the test sample titer was calculated according to the following formula 1:

[0104] Test sample titer (IU / mL) = Pr x (Ds x Es) / (Dr x Er) (Formula 1)

[0105] Wherein: Pr is the biological activity (titer) of the standard sample (herein refers to the national standard sample for the determination of the biological activity of recombinant human IL-2), IU / mL; Ds is the pre-dilution fold of the test sample (herein refers to the fusion protein ABD-Fc-IL-2); Dr is the pre-dilution fold of the standard sample; Es is the dilution fold of the test sample corresponding to the half-effective amount of the standard sample; and Er is the dilution fold of the half-effective amount (EC 50 ) of the standard sample.

[0106] The results are shown in Figure 2 and 3 , wherein Figure 2 The four-parameter regression fitting curve (log Ig activity-OD 570 value) of the standard sample of recombinant human IL-2 is shown, Figure 3 The four-parameter regression fitting curve (Ig dilution fold-OD 570 value) of the fusion protein ABD / Fc / IL-2 and the standard sample of recombinant human IL-2 is shown. Referring to the fitting curve formula in Figure 2 and by the above formula 1, the titer of the test sample fusion protein ABD-Fc-IL-2 is calculated to be about 3.29 x 10 7 IU / mL.

[0107] The specific activity of the test sample (fusion protein ABD-Fc-IL-2) is calculated as the ratio of the activity titer of the test sample obtained by the above formula 1 to its protein concentration, i.e. the specific activity, which is IU / mg. Therefore, the specific activity of the fusion protein ABD-Fc-IL-2 provided by the present application is calculated to be about 3.29 x 10 8 IU / mg, which is higher than 1 x 10 8 IU / mg, and according to the results shown in Figure 3 , the specific activity of the fusion protein ABD-Fc-IL-2 is significantly higher than that of the standard sample of recombinant IL-2 (about 8.5 x 10 6IU / mg) is also obviously higher than the specific activity of the fusion protein IL-2 / HSA (the highest is 1.026 x 10 7 IU·mg -1 ).

[0108] Example 3: Serum albumin binding test of fusion protein ABD / Fc / IL-2

[0109] This example uses the Pull down / Western blot method to detect the binding of the fusion protein ABD / Fc / IL-2 expressed and purified in Example 1 above to serum albumin, and specifically includes the following steps.

[0110] 1) Test design:

[0111] 100 ng of ABD-Fc-IL-2 and 20 μL of Protein A gel are added to Group A;

[0112] 100 ng of human serum albumin HSA and 20 μL of Protein A gel are added to Group B;

[0113] 100 ng of ABD-Fc-IL-2, 100 ng of human serum albumin HSA, and 20 μL of Protein A gel are added to Group C.

[0114] 2) The mixtures in each group are incubated at 4°C for 4 hours, washed with PBST to remove unbound proteins, and subjected to western blot detection.

[0115] The results, as shown in Figure 4 , show that the fusion protein ABD / Fc / IL-2 provided by the present application can bind to human serum albumin (HSA) through the ABD therein, and this binding property can be used to modify the fusion protein with serum albumin having a longer in vivo circulation half-life, thereby prolonging the in vivo plasma circulation half-life of the fusion protein without the need to introduce human serum albumin with a larger molecular weight into the fusion protein, which is more conducive to the industrial production of the fusion protein and is also more suitable for clinical application due to easier penetration of tissues in the living body.

[0116] Example 4: Dynamic detection of blood drug concentration of fusion protein ABD / Fc / IL-2

[0117] This example detects the mouse pharmacokinetics of the fusion protein ABD / Fc / IL-2 obtained by expression and purification in Example 1 above to determine that the fusion protein ABD / Fc / IL-2 indeed has a prolonged in vivo circulation half-life, and specifically includes the following steps.

[0118] 1) 5-6 weeks old BalB / c female mice were selected, Fc / IL-2 (fusion protein obtained by connecting full-length human immunoglobulin Fc segment and IL-2 according to the same recombinant technology as in Example 1 and expressed by CHO cells) was used as a control group, and fusion protein ABD / Fc / IL-2 was used as a test group. The control group and the test group were set up into low (250 μg / kg, the dose of the fusion protein, the same below), medium (500 μg / kg), and high (1000 μg / kg) three dose groups, and subcutaneous injection was given on the back of the mice, and tail blood was collected at 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after injection, respectively, and serum was separated. The concentration of Fc / IL-2 and fusion protein ABD / Fc / IL-2 in the serum was detected respectively to detect the change of the concentration with time.

[0119] 2) The concentration of fusion protein ABD / Fc / IL-2 and Fc / IL-2 was detected by double antibody sandwich IL-2 ELISA kit, wherein the fusion protein ABD / Fc / IL-2 and Fc / IL-2 were diluted by 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9 pg / ml, etc. ratio, then ELISA detection was carried out and standard curve was drawn. The specific steps of ELISA detection were as follows: (1) standard and sample (serum sample) were set up into two duplicate wells, 100 microliters were added, and 37℃ incubation was carried out for 90 minutes, and then the plate was washed 4 times. (2) 100 microliters of biotinylated antibody was added to each well, 37℃ incubation was carried out for 60 minutes, and the plate was washed 4 times; (3) 100 microliters of enzyme conjugate was added, 37℃ incubation was carried out for 30 minutes, and the plate was washed 5 times; (4) 50 microliters of color developing substrate solution was added, 37℃ incubation was carried out for 15 minutes, and then the reaction was terminated, and OD value was detected. Graphpad Prism 9 software was used for drawing and analysis, and Student's t test was used for comparison between two groups. 450

[0120] The concentration of fusion protein Fc / IL-2 and fusion protein ABD / Fc / IL-2 in serum changed with time as follows: Figure 5 ​As shown in the figure, wherein A, B and C represent the pharmacokinetic curves of mice in low (250 μg / kg), medium (500 μg / kg) and high (1000 μg / kg) dose groups respectively, it can be seen that in the dose range of 0.25-1 mg / kg, both the fusion protein ABD-Fc-IL-2 and Fc-IL-2 show nonlinear pharmacokinetic characteristics, and compared with the pharmacokinetic characteristics of the fusion protein Fc / IL-2 (the peak time of blood concentration after subcutaneous injection is about 2.5 hours, and the significant biological activity cannot be detected about 20 hours after administration, and the plasma circulation half-life is about 2.5 hours), the fusion protein ABD-Fc-IL-2 obviously shows the characteristics of longer in vivo action time (the peak time of blood concentration after subcutaneous injection is about 8 hours, the significant biological activity can still be detected 48 hours after administration, and the plasma circulation half-life is about 10 hours) and lower clearance rate. Therefore, the fusion protein ABD-Fc-IL-2 provided by the present application can prolong the plasma circulation half-life of IL-2 in vivo, which is beneficial to ensure stable IL-2 plasma concentration.

[0121] The toxic side effects of the existing IL-2 products and the fusion proteins provided in the above documents 1 to 4 are mainly caused by the binding of IL-2 to the α receptor and / or β receptor, therefore, after replacing the natural IL-2 in the fusion protein ABD-Fc-IL-2 (wild type) expressed and purified in the above Example 1 with the mutants of IL-2 (including, for example, IL-2v1, IL-2v2 and IL-2v3), a fusion protein with low toxic side effects is also obtained, and the fusion protein also has the same high biological activity, long plasma circulation half-life and pharmacokinetic characteristics as the fusion protein ABD-Fc-IL-2 expressed and purified in the above Example 1, which will not be described here.

[0122] Finally, it should be noted that the above description is only for the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Beijing University Sun Hongyan <120> A fusion protein ABD / Fc / IL-2, a coding gene thereof, a preparation method and an application <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 56 <212> PRT <213> Artificial Sequence <400> 1 Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val 1 5 10 15 Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys 20 25 30 Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile 35 40 45 Asp Glu Ile Leu Ala Ala Leu Pro 50 55 <210> 2 <211> 231 <212> PRT <213> Artificial Sequence <400> 2 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro 115 120 125 Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly 225 230 <210> 3 <211> 133 <212> PRT <213> Artificial Sequence <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 4 <211> 133 <212> PRT <213> Artificial Sequence <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 133 <212> PRT <213> Artificial Sequence <400> 5 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Ala Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 6 <211> 133 <212> PRT <213> Artificial Sequence <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Ala Ala Gin Ser Lys Asn Phe His Phe 65 70 75 80 Asp Pro Arg Asp Val Val Ser Asn lie Asn Val Phe Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ala Gin Ser lie 115 120 125 lie Ser Thr Leu Thr 130 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <400> 7 Gly Gly Ser 1 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 9 <211> 438 <212> PRT <213> Artificial Sequence <400> 9 Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val 1 5 10 15 Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys 20 25 30 Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile 35 40 45 Asp Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Glu Pro Lys Ser Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 85 90 95 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 195 200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 290 295 300 Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu 305 310 315 320 His Leu Leu Leu Asp Leu Gin Met Ile Leu Asn Gly Ile Asn Asn Tyr 325 330 335 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro 340 345 350 Lys Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu 355 360 365 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His 370 375 380 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 385 390 395 400 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 405 410 415 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser 420 425 430 Ile Ile Ser Thr Leu Thr 435 <210> 10 <211> 1314 <212> DNA <213> Artificial Sequence <400> 10 cagcatgacg aggcagtcga cgcaaattcc ctggccgagg caaaggtcct ggcaaataga 60 gaactggata agtacggggt gtccgattac tataagaacc tgatcaacaa tgccaagacc 120 gtggagggcg tgaaggctct gatcgacgag atcctggccg ctctgccagg aggcagcgag 180 ccaaagtcca gcgataagac ccacacatgc ccaccttgtc cagctccaga gctgctgggc 240 ggaccttccg tgttcctgtt tccacccaag ccaaaggaca ccctgtacat cacaagggag 300 cctgaggtga cctgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 360 tggtacgtgg atggcgtgga ggtgcataat gctaagacaa agcctagaga ggagcagtac 420 tggtacgtgg atggcgtgga ggtgcataat gctaagacaa agcctagaga ggagcagtac 420aactccacct atcgcgtggt gagcgtgctg acagtgctgc atcaggactg gctgaacggc 480 aaggagtaca agtgcaaggt gtctaataag gccctgcctg ctccaatcga gaagaccatc 540 tccaaggcta agggacagcc cagggagcct caggtgtaca cactgcctcc atcccgggag 600 gagatgacca agaaccaggt gagcctgaca tgtctggtga agggcttcta tcccagcgac 660 atcgctgtgg agtgggagtc taatggccag cctgagaaca attacaagac cacaccccct 720 gtgctggaca gcgatggctc tttctttctg tattctaagc tgaccgtgga taagtccaga 780 tggcagcagg gcaacgtgtt ttcttgttcc gtgatgcacg aggccctgca caatcattat 840 acacagaaga gcctgtctct gtccccagga ggaggaggag gctccggcgg aggaggcagc 900 ggcggcggcg gatccgctcc cacctcttcc agcaccaaga agacacagct gcagctggag 960 catctgctgc tggatctgca gatgatcctg aacggcatca acaattacaa gaatccaaag 1020 ctgacccgca tgctgacatt caagttttat atgcccaaga aggccaccga gctgaagcac 1080 ctgcagtgcc tggaggagga gctgaagccc ctggaggagg tgctgaacct ggctcagtct 1140 aagaatttcc atctgaggcc tcgggacctg atctccaaca tcaatgtgat cgtgctggag 1200 ctgaagggca gcgagacaac cttcatgtgc gagtacgccg atgagacagc tacaatcgtg 1260 gagttcctga atcgttggat caccttcgca cagagcatca tctcaaccct gacc 1314 <210> 11 <211> 438 <212> PRT <213> Artificial Sequence <400> 11 Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val 1 5 10 15 Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys 20 25 30 Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile 35 40 45 Asp Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Glu Pro Lys Ser Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 85 90 95 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 195 200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Gin Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 290 295 300 Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu 305 310 315 320 His Leu Leu Leu Asp Leu Gin Met Ile Leu Asn Gly Ile Asn Asn Tyr 325 330 335 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro 340 345 350 Lys Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu 355 360 365 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His 370 375 380 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 385 390 395 400 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 405 410 415 Ala Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Ala Gin Ser 420 425 430 lie lie Ser Thr Leu Thr 435 <210> 12 <211> 438 <212> PRT <213> Artificial Sequence <400> 12 Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val 1 5 10 15 Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys 20 25 30 Asn Leu lie Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu lie 35 40 45 Asp Glu lie Leu Ala Ala Leu Pro Gly Gly Ser Glu Pro Lys Ser Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 85 90 95 lie Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser 195 200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 290 295 300 Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 305 310 315 320 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 325 330 335 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro 340 345 350 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 355 360 365 Lys Pro Leu Glu Glu Val Leu Asn Ala Ala Gln Ser Lys Asn Phe His 370 375 380 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 385 390 395 400 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 405 410 415 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser 420 425 430 Ile Ile Ser Thr Leu Thr 435 <210> 13 <211> 438 <212> PRT <213> Artificial Sequence <400> 13 Gln His Asp Glu Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val 1 5 10 15 Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys 20 25 30 Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile 35 40 45 Asp Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Glu Pro Lys Ser Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 85 90 95 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser 195 200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 290 295 300 Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 305 310 315 320 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 325 330 335 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro 340 345 350 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 355 360 365 Lys Pro Leu Glu Glu Val Leu Asn Ala Ala Gln Ser Lys Asn Phe His 370 375 380 Phe Asp Pro Arg Asp Val Val Ser Asn Ile Asn Val Phe Val Leu Glu 385 390 395 400 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 405 410 415 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser 420 425 430 Ile Ile Ser Thr Leu Thr 435

Claims

1. A fusion protein ABD / Fc / IL-2, characterized in that, The amino acid sequence of the fusion protein ABD / Fc / IL-2 is the sequence shown in SEQ ID NO:

9.

2. The gene encoding the fusion protein ABD / Fc / IL-2 according to claim 1, wherein the nucleotide sequence is the nucleotide sequence encoding SEQ ID NO:

9.

3. The encoding gene according to claim 2, wherein the nucleotide sequence is as shown in SEQ ID NO:

10.

4. A recombinant expression vector for expressing the fusion protein ABD / Fc / IL-2 of claim 1, characterized in that, The recombinant expression vector contains the coding gene as described in claim 2 or 3.

5. A transgenic cell line for expressing the fusion protein ABD / Fc / IL-2 of claim 1, characterized in that, The transgenic cell line contains the coding gene as described in claim 2 or 3 or the recombinant expression vector as described in claim 4.

6. A method for recombinantly expressing the fusion protein ABD / Fc / IL-2 of claim 1, comprising the following steps: 1) Construct the transgenic cell line as described in claim 5, and culture the transgenic cell line; 2) The protein is isolated and purified from the culture medium or cells of the transgenic cell line to obtain the fusion protein ABD / Fc / IL-2.

7. The method according to claim 6, wherein the transgenic cell line is a CHO cell line.

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