CN115232215A - Fusion protein ABD/Fc/IL-2 and encoding gene, preparation method and application thereof - Google Patents

Abstract

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

Description

A kind of fusion protein ABD/Fc/IL-2 and its encoding gene, preparation method and application

technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to a fusion protein (named as fusion protein ABD/Fc/IL- 2) and its encoding gene, preparation method and application, especially involving both high biological activity (protein specific activity higher than 1×10 ⁸ IU/mg) and long plasma circulating half-life (about 10 hours) or both Clinical application of the fusion protein ABD/Fc/IL-2 with high biological activity, long plasma circulating half-life and low toxicity and side effects in the preparation of medicines for the treatment of malignant tumors, infectious diseases and low immunity of the body.

Background technique

Interleukin-2 (IL-2) is a traditional immune-enhancing cytokine. Clinical practice has proved that IL-2 can effectively stimulate the proliferation of various immune cells such as cytotoxic T cells and NK cells, and enhance the body's immunity; it can also exert more lasting tumor immune protection by inducing the body's immune memory function, effectively delaying the postoperative period. Tumor recurrence, inhibition of tumor growth, combined therapy with immune checkpoint blockers and other drugs, and synergistic effect of tumor therapy have been widely used in the comprehensive treatment of renal cancer, melanoma, colon cancer, lung cancer and other malignant tumors. Treatment of infectious diseases such as AIDS and viral hepatitis.

However, existing IL-2 products (such as recombinant IL-2) have the following major drawbacks, which limit their clinical application: (1) IL-2 has a very short plasma half-life (in vivo circulating half-life of only 6.9 minutes), so in order to achieve Effective plasma concentrations require frequent dosing, resulting in poor compliance and difficulty in ensuring stable plasma concentrations of IL-2, while low doses of IL-2 induce regulatory T cells, suppress immune responses, and promote tumor escape (2) The recombinant IL-2 products expressed by bacteria are renatured from inclusion bodies, and their specific protein activity (unit activity) is low, making it difficult to achieve the expected immune enhancement and regulation mediated by IL-2. Effect.

At present, there is no long-acting IL-2 product on the market at home and abroad. The products under development mainly use technologies such as recombinant fusion of human serum albumin (HSA) or immunoglobulin Fc segment with IL-2 to improve the plasma circulating half-life of IL-2.

Patent document CN1884520A (hereinafter referred to as document 1) discloses a fusion protein of human interleukin-2 and human serum albumin (IL-2/HSA), which uses recombinant technology to connect human IL-2 and HSA and express it in Pichia pastoris. Medium and high-efficiency expression is obtained, mainly using human serum albumin with a long plasma half-life (about 20 days) to modify IL-2 macromolecularly to achieve long-term IL-2 action (subcutaneous injection of IL-2/HSA fusion protein) The peak time of plasma concentration after administration 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, in this document 1, using bacteria (Pichia pastoris) to express the fusion protein IL-2/HSA, the specific activity (unit activity) of the fusion protein IL-2/HSA is not high (about 1.147×10 ⁶ IU/mg , see e.g. Document 2), it may be difficult to achieve the expected immune enhancement and regulation mediated by IL-2, and when the fusion protein is expressed in Pichia pastoris, the product may have heterogeneity, excessive glycosylation and degradation and other problems, and may lead to the increase of the immunogenicity of the fusion protein, which affects the efficacy of multiple clinical administrations, and is greatly limited in production and application. Although patent document CN104789594A (hereinafter referred to as document 3) has constructed a CHO cell line that stably and efficiently expresses human serum albumin and interleukin II fusion protein (IL-2/HSA), the expressed fusion protein is closest to the natural protein, and it is very It secretes less of its own endogenous protein, and the product is secreted extracellularly, which is beneficial for purification, but the specific activity of the fusion protein obtained by expression and purification in this document 3 is still not high (the specific activity of the IL-2-HSA fusion protein is 8.3×10 ⁶ IU·mg ^-1 , the specific activity of HSA-IL-2 fusion protein is 1.026×10 ⁷ IU·mg ^-1 ), and it may 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 documents 1, 2 and 3 has a relatively large molecular weight (about 82KDa), which is not conducive to tissue penetration in vivo, and may also limit its clinical efficacy and application value.

Patent document CN102174111A (hereinafter referred to as document 4) discloses a human interleukin-2-Fc fusion protein (IL-2/Fc), which is obtained by using recombinant technology to connect human IL-2 and IgG Fc and express it in CHO cells. The reversible binding of the Fc in the IL-2/Fc fusion protein to the FcRn receptor in the tissue is used to increase its tissue stability, thereby prolonging the action time of IL-2 (according to the description of the document 4, Figure 6 is calculated and obtained). The actual plasma circulating half-life of the fusion protein IL-2/Fc is about 2.5 hours), and the fusion protein provided by the document 4 also has ADCC and CDC effects due to the Fc segment in the fusion protein. However, although the fusion protein IL-2/Fc provided in this document 4 can prolong its circulating half-life in vivo compared with the existing recombinant IL-2 (recombinant human interleukin-2 (125Ser)/interleukin-2 for injection), However, the prolongation effect is still insufficient, and its clinical application is greatly limited. On the other hand, the fusion protein IL-2/Fc disclosed in this document 4 has only the same affinity for binding to IL-2 receptor as recombinant IL-2, so its biological activity (mainly protein specific activity) is low. , it may also be difficult to achieve the expected immune enhancement and regulation mediated by IL-2.

SUMMARY OF THE INVENTION

In view of one or more problems in the prior art, one aspect of the present invention provides a fusion protein ABD/Fc/IL-2, which comprises human serum albumin binding domain ABD (comprising SEQ ID NO: 1), full-length human immunoglobulin Fc segment and IL-2 (comprising the amino acid sequence 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 (protein specific activity is higher than 1×10 ⁸ IU/mg); the full-length human immunoglobulin Fc segment is selected from human IgG1, IgG2, IgG3 and IgG4.

The above-mentioned full-length human immunoglobulin Fc segment comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

The above-mentioned mutant of IL-2 is a mutation obtained by mutating any one or more of positions 42, 45, 72, 80, 81, 85, 86 and 92 of the amino acid sequence of native IL-2 body;

Preferably, F42, Y45, L72, L80, R81, L85, I86, and I92 of the amino acid sequence of native IL-2 are mutated to A42, A45, A72, F80, D81, V85, V86, and F92, respectively;

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 native IL-2 F42 and Y45 of the amino acid sequence of IL-2v1 are mutated into A42 and A45 respectively; the mutant IL-2v2 is the mutant IL-2v1 whose amino acid sequence L72 is mutated to A72; the mutant IL-2v3 is the mutant IL-2v2 L80, R81, L85, I86, and I92 of the amino acid sequence were mutated to F80, D81, V85, V86, and F92, respectively;

Further preferably, the mutant of IL-2 comprises the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in the sequence listing.

The above-mentioned fusion protein ABD/Fc/IL-2 also comprises a connecting peptide or an analog thereof for connecting the human serum albumin binding domain ABD, the full-length human immunoglobulin Fc segment and IL-2 or a mutant thereof into a The fusion protein ABD/Fc/IL-2;

Preferably, the linking peptide or its analog is used for linking the carboxy terminus of the human serum albumin binding domain ABD and the amino terminus of the Fc segment of a full-length human immunoglobulin, and for linking the full-length human immunoglobulin The carboxyl terminus of the protein Fc segment and the amino terminus of IL-2 or its mutants;

Optionally, the amino acid residue sequence of the linking peptide is shown in SEQ ID NO: 7 or SEQ ID NO: 8 in the sequence listing.

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

1) the amino acid residue sequence shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing;

2) The amino acid residue sequence shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing is subjected to substitution, deletion or addition of one to ten amino acid residues and the Amino acid sequence with high biological activity (specific protein activity higher than 1×10 ⁸ IU/mg).

Another aspect of the present invention provides a gene encoding the above-mentioned fusion protein ABD/Fc/IL-2 (ABD/Fc/IL-2), the nucleotide sequence of which is one of the following nucleotide sequences:

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

2) the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing;

3) The same as the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing A nucleotide sequence with more than 90% homology and high biological activity (protein specific activity higher than 1×10 ⁸ IU/mg) after the expressed protein;

4) Under high stringency conditions, it can be compared with the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID in the coding sequence listing. Nucleotide sequence to which the nucleotide sequence of NO: 13 hybridizes.

Another aspect of the present invention also provides a recombinant expression vector pcDNA3.1-ABD/Fc/IL-2, which comprises the above-mentioned encoding gene (ABD/Fc/IL-2) for expressing the above-mentioned fusion protein ABD/Fc /IL-2.

Another aspect of the present invention also provides a transgenic cell line or host bacteria, which comprises the above-mentioned encoding gene (ABD/Fc/IL-2) or the above-mentioned recombinant expression vector pcDNA3.1-ABD/Fc/IL-2, using for expressing the above fusion protein ABD/Fc/IL-2.

Another aspect of the present invention provides a medicine for treating malignant tumors, infectious diseases and low immunity, the medicine comprising the above fusion protein ABD/Fc/IL-2 or encoding gene (ABD/Fc/IL-2) -2) as an active ingredient;

Optionally, the medicament further comprises one or more of the following: pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, Surfactant.

Another aspect of the present invention also provides a method for recombinantly expressing the above-mentioned fusion protein ABD/Fc/IL-2, comprising the following steps:

1) construct above-mentioned transgenic cell line or host bacterium, and cultivate this transgenic cell line or host bacterium;

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

Preferably, the method specifically includes the following steps:

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

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

c) Purify the fusion protein ABD/Fc/IL-2 by affinity chromatography or ion exchange chromatography.

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

The fusion protein ABD/Fc/IL-2 provided based on the above technical solution is composed of human serum albumin binding domain ABD, human immunoglobulin Fc segment (full length) and interleukin 2 (IL-2) three-part fusion (can be found in The carboxyl terminus of different parts and the amino acid can also be directly fused by linking peptides or their analogs), wherein ABD can be combined with human serum albumin, and then use the modification of human serum albumin with a longer plasma half-life to make the fusion. The protein ABD/Fc/IL-2 has a longer action time; on the one hand, the human immunoglobulin Fc segment can increase the tissue stability of the fusion protein ABD/Fc/IL-2 by reversibly binding to the FcRn receptor in the tissue On the other hand, the fusion protein ABD/Fc/IL-2 can also have CDC and ADCC effects, thereby exerting immune effector functions; IL-2 can effectively stimulate the proliferation of various immune cells such as cytotoxic T cells and NK cells, thereby increasing the The body's basic immunity.

Compared with the prior art, the present invention has the following beneficial effects:

1) The fusion protein ABD/Fc/IL-2 provided by the present invention has higher biological activity (protein specific activity is about 3.29×10 ⁸ IU/mg), which is significantly higher than that disclosed in the above-mentioned documents 1, 2 and 3. The biological activity of the fusion protein IL-2/HSA (the highest protein specific activity was 1.026×10 ⁷ IU·mg ^-1 ) and the biological activity of the recombinant IL-2 standard (the protein specific activity was about 8.5×10 ⁶ IU/mg, Please refer to document 3); also significantly higher than the biological activity of the fusion protein IL-2/Fc disclosed in the above document 4 (it has the same affinity for binding to IL-2 receptor as recombinant IL-2, which is reflected in the protein In terms of specific activity, it is equivalent to the protein specific activity of recombinant IL-2 (about 8.5×10 ⁶ IU/mg), so the fusion protein ABD/Fc/IL-2 provided by the present invention can be easier compared to the prior art To achieve the expected effect of immune enhancement and regulation mediated by IL-2.

2) The fusion protein ABD/Fc/IL-2 provided by the present invention can significantly prolong the in vivo action time of IL-2 (the peak time of the blood drug concentration of subcutaneous injection is about 8 hours, and it can still be detected after 48 hours of administration; It has obvious biological activity, and 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 the above documents 1, 2 and 3), and is significantly longer than the fusion protein IL-2/HSA disclosed in the above document 4. The plasma circulating half-life of Fc (according to Fig. 6 recorded in Document 4, the plasma circulating half-life is actually about 2.5 hours, and according to the description in Fig. 5 of the present specification, it can also be calculated that its plasma circulating half-life is about 2.5 hours, which is similar to that of the document. 4) and the in vivo plasma circulating half-life of the existing recombinant IL-2 (6.9 minutes), thereby prolonging the in vivo plasma circulating half-life of IL-2, which is beneficial to ensure a stable IL-2 plasma concentration.

3) The full length of the fusion protein ABD/Fc/IL-2 provided by the present invention is 438 amino acids, and its molecular weight is relative to the fusion protein IL-2/HSA (full length of about 718 amino acids) provided in the above-mentioned documents 1, 2 and 3. amino acid) is much smaller, and the molecular weight of ABD in the fusion protein ABD/Fc/IL-2 provided by the present invention is about 6KDa, which is also higher than that in the fusion protein IL-2/HSA provided in the above-mentioned documents 1, 2 and 3. The molecular weight (about 66.5KDa) of HSA with similar functions is much smaller, so it is more conducive to penetrating tissues in vivo, and is conducive to expression and production, and is more conducive to industrial production and clinical application.

4) The fusion protein ABD/Fc/IL-2 provided by the present invention can be expressed and produced by CHO cells, and the production process is simple, which not only makes its structure closest to the natural protein spatial conformation, but also can avoid the use of bacteria such as Pichia pastoris to the greatest extent. The product may have problems such as inhomogeneity, excessive glycosylation and degradation during production, thereby avoiding the increase of immunogenicity of the product fusion protein.

5) The present invention 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, which is not only a It has the advantages of high biological activity and long plasma circulating half-life of the fusion protein ABD/Fc/IL-2 (wild type), and is also compared to the fusion protein IL disclosed in the wild-type IL-2, the above-mentioned documents 1, 2 and 3. -2/HSA and the fusion protein IL-2/Fc disclosed in the above-mentioned document 4 have the advantage of low toxicity, so it can avoid strong toxic side effects caused by high doses of IL-2, such as vascular leak syndrome (VLS, causing The accumulation of water in human organs, such as pulmonary edema and liver cell damage), makes it more suitable for clinical use in the preparation of drugs for the treatment of malignant tumors, infectious diseases, low immunity and other diseases.

To sum up, the fusion protein ABD/Fc/IL-2 provided by the present invention not only integrates the functions of serum albumin, immunoglobulin Fc segment and IL-2, but also endows the fusion protein ABD/Fc/IL-2 with long-term effects. The plasma circulating half-life, CDC and ADCC effects and IL-2 can effectively stimulate the proliferation of cytotoxic T cells, NK cells and other immune cells, thereby improving the body's basic immunity, and also significantly improve the fusion protein ABD/Fc/ The biological activity of IL-2 (protein specific activity is higher than 1×10 ⁸ IU/mg) can even reduce the toxic and side effects of the fusion protein ABD/Fc/IL-2. Therefore, the fusion protein ABD/Fc/IL-2 provided by the present invention is more suitable for clinical application, and its immune ability and immune effect are also better than IL-2 related products existing in the prior art.

Description of drawings

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

Fig. 2 is the four-parameter regression fitting curve of recombinant human IL-2 standard product;

Fig. 3 is the four-parameter regression fitting curve of fusion protein ABD/Fc/IL-2 provided by an embodiment of the present invention;

Fig. 4 is the Pull down/Westernblot detection result of fusion protein ABD/Fc/IL-2 provided by an embodiment of the present invention;

Fig. 5 is the mouse pharmacokinetic curve of fusion protein ABD/Fc/IL-2 and fusion protein Fc/IL-2 provided by an embodiment of the present invention, wherein A, B and C represent three different dose groups, respectively Pharmacokinetic profiles in mice.

Detailed ways

The present invention aims to provide a fusion protein ABD/Fc/IL-2 (wild type) with both high biological activity and long plasma circulating half-life for the clinical application of IL-2, and further provides a A fusion protein ABD/Fc/IL-2 (mutant type) with excellent biological activity, long plasma circulating half-life and low toxicity and side effects, and provides an industrialized production method of the fusion protein, so that IL-2 can play a better role In the treatment of malignant tumors, infectious diseases, low immunity and other diseases. The present invention is specifically realized in the following manner.

One object of the present invention is to provide a fusion protein with both high biological activity and long plasma circulating half-life, named as fusion protein ABD/Fc/IL-2 (wild type), which comprises a human serum albumin binding domain (ABD), full-length immunoglobulin Fc fragment and human IL-2 (wild-type), which are relative to IL-2 related products existing in the prior art (eg recombinant IL-2 or those disclosed in documents 1 to 4 above) IL-2 fusion protein) has significantly higher biological activity and long plasma circulating half-life, and all retain the ability of IL-2 to stimulate the proliferation of immune cells such as T cells and NK cells, and can exert both CDC and ADCC effects.

Specifically, the fusion protein ABD/Fc/IL-2 (wild type) is connected to ABD at the amino terminus (N-terminus) of the immunoglobulin Fc segment and at the carboxyl terminus (C-terminus) of the Fc segment through a linking peptide IL-2 fusion protein. 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 both high biological activity and long plasma circulating half-life. Among them: ABD can be combined with serum albumin in the blood circulation, so that the serum albumin with a longer in vivo circulating half-life can carry out macromolecular modification of the fusion protein, and prolong the in vivo plasma circulation half-life of the fusion protein. (For example, the above-mentioned documents 1, 2 and 3) use serum albumin with a larger molecular weight to fuse with IL-2 to achieve the purpose of prolonging the half-life of plasma circulation in vivo, thereby significantly reducing the molecular weight of the fusion protein, which is convenient for expression and production, and It is easier to penetrate tissues in vivo; on the one hand, the immunoglobulin Fc segment can increase the tissue stability of the fusion protein by reversibly binding to FcRn in the tissue, and on the other hand, it can exert the immune effects of CDC and ADCC, thereby enhancing the immunity of the fusion protein. Efficacy; 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 to improve the body's basic immunity. Therefore, the fusion protein ABD/Fc/IL-2 (wild type) provided by the present invention integrates the functions of serum albumin, immunoglobulin Fc segment and IL-2, and also has a smaller molecular weight, which is more conducive to production and more suitable for The advantages of clinical use, and at the same time have the advantages of higher biological activity (protein specific activity) compared to 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 invention is more suitable for clinical production and application.

Preferably, the selection of immunoglobulin Fc segment includes various sources such as IgG1, IgG2, IgG3 and IgG4, and the fusion protein ABD/Fc/IL-2 (wild type) can be directly fused by ABD, Fc segment and IL-2. For example, the carboxyl terminus of ABD is directly fused to the amino terminus of the Fc segment, the carboxyl terminus of the Fc segment is directly fused to the amino terminus of IL-2, or it can be fused through a linker peptide or its analogs, and the choice of linker peptide can be multiple Various, for example, the linker peptide of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 in the sequence listing or its analog can be selected. ABD and Fc can also be replaced by their analogs, and the differences between these analogs and ABD and Fc can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagenic agents, but also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids.

Specifically, the human serum albumin binding domain ABD comprises the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and the full-length human immunoglobulin Fc segment comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing The amino acid sequence shown in 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:

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

2) The amino acid residue sequence shown in SEQ ID NO: 9 in the sequence listing is subjected to substitution, deletion or addition of amino acid residues and has both serum albumin specificity, Fc function and long-term stimulation/activation of immune cells. .

Wherein, SEQ ID NO: 9 consists of 438 amino acid residues, the amino acid residues at the 1-56 (56aa) positions from the amino terminal are ABD, and the amino acid residues at the 57-59 (3aa) positions from the amino terminal are connecting peptides, The amino acid residue at the 60-290 (231aa) position from the amino terminal is the immunoglobulin Fc segment, the amino acid residue at the 291-305 (15aa) position from the amino terminal is the connecting peptide, and the amino terminal position 306-438 (133aa) The amino acid residue is IL-2.

Further, on the one hand, polypeptide fragments, derivatives and analogs of the fusion protein ABD/Fc/IL-2 (wild type) also belong to the present invention, which are combined with the fusion protein ABD/Fc/IL-2 (wild type). ) have the same biological function or activity, wherein a polypeptide fragment is defined as: 1) a polypeptide substituted by one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues Residues may or may not be encoded by the genetic code; 2) polypeptides with substitution groups in one or more amino acid residues; 3) polypeptides formed by fusion of mature polypeptides with another compound;

4) A polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a sequence used to purify this polypeptide, or a fusion protein of an antibody fragment or other antigenic ligand sequence), or a nucleic acid sequence that will encode another polypeptide The coding sequence of the fusion polypeptide can be obtained by fusing (or part thereof) with the nucleic acid sequence (or part thereof) of the present invention, and then the coding sequence of the fusion polypeptide can be expressed to produce the fusion polypeptide. Such techniques for producing fusion polypeptides are well known in the art and include ligating the coding sequences encoding the polypeptides so that they are in the same reading frame and allowing expression of the fusion polypeptides to be controlled by the same promoter and terminator.

Derivatives of fusion protein ABD/Fc/IL-2 (wild type) refer to: (1) fusion proteins constructed by using IgG Fc segments or IL-2 mutants from other sources in the present invention, (2) using the present invention It is a fusion protein constructed by ABD/Fc and other cytokines (such as IL-12, IL-15, etc.) that can stimulate the proliferation of immune cells.

The difference between the analog of fusion protein ABD/Fc/IL-2 (wild type) and fusion protein ABD/Fc/IL-2 (wild type) can be the difference in amino acid sequence, or it can be modified form that does not affect the sequence. differences, or both. Such analogs include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagenic agents, but also by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids. It should be understood that the amino acid residue sequences of the fusion proteins of the present invention are not limited to the representative sequences exemplified above.

Further, on the other hand, the fusion protein ABD/Fc/IL-2 (wild type) can also be modified ABD/Fc/IL that has been modified to improve its anti-proteolytic properties or optimize its solubility properties -2 polypeptides. Modified (usually without altering primary structure) forms include: 1) chemically derivatized forms of the polypeptide in vivo or in vitro, such as acetylation or carboxylation; 2) glycosylation, such as those in the synthesis and processing of the polypeptide or further processing steps Polypeptides produced by glycosylation modification in Sequence of amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine).

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

Clinical evidence shows that although higher doses of IL-2 are used, the better the anti-tumor effect, but high doses of IL-2 can cause strong side effects, such as vascular leak syndrome (VLS), which leads to internal organs in the human body. Accumulation of fluid, such as pulmonary edema and liver cell damage. Therefore, for IL-2 products, the bottleneck factors that limit their clinical application are also the large toxic and side effects. Reducing toxicity will be more conducive to the clinical application of IL-2 related products. The inventors replaced the native IL-2 in the fusion protein ABD/Fc/IL-2 (wild type) provided by the present invention with a key site (with the alpha and/or IL-2) based on the methods disclosed in the patent document CN104231068A A low-toxicity IL-2 mutant obtained by amino acid mutation of β-receptor or binding point of β receptor), thereby providing a fusion protein with high biological activity, long plasma circulating half-life and low toxicity. Specifically, the low toxicity IL-2 mutant can occur at any one or more of positions 42, 45, 72, 80, 81, 85, 86, and 92 of the amino acid sequence of native IL-2. Mutants obtained after mutation; preferably, F42, Y45, L72, L80, R81, L85, I86, I92 of the amino acid sequence of natural IL-2 are mutated to A42, A45, A72, F80, D81, V85, V86, F92; further preferably, the low toxicity IL-2 mutants include the following three types: (1) Interleukin 2 mutant 1 (IL-2v1): the difference between its amino acid sequence and the amino acid sequence of natural IL-2 is: natural F42 and Y45 of the amino acid sequence of IL-2 are mutated into A42 and A45 respectively in the amino acid sequence of IL-2v1, and the amino acid residues of this IL-2v1 are shown in SEQ ID NO: 4 in the sequence table. At this time, the fusion protein ABD The amino acid sequence of /Fc/IL-2 (mutant type) is shown in SEQ ID NO: 11 in the sequence listing; (2) Interleukin 2 mutant 2 (IL-2v2): its amino acid sequence is the same as that of IL-2v1 The difference of the sequence is: L72 of the IL-2v1 amino acid sequence is mutated to A72 in the IL-2v2 amino acid sequence, and the amino acid residue of the IL-2v2 is shown in SEQ ID NO: 5 in the sequence table. At this time, the fusion protein ABD/Fc The amino acid sequence of /IL-2 (mutant type) is shown in SEQ ID NO: 12 in the sequence listing; (3) Interleukin 2 mutant 3 (IL-2v3): its amino acid sequence is the same as that of IL-2v2 The difference is: L80, R81, L85, I86, and I92 in the amino acid sequence of IL-2v2 are mutated into F80, D81, V85, V86, and F92, respectively, in the amino acid sequence of IL-2v3. The amino acid residues of IL-2v3 are as follows. SEQ ID NO: 6 in the list shows the amino acid sequence of the fusion protein ABD/Fc/IL-2 (mutant type) at this time as SEQ ID NO: 13 in the sequence list. By mutating key sites of native IL-2, the affinity of alpha receptors and IL-2 can be reduced or the affinity of IL-2 and beta receptors can be enhanced, thereby affecting its ability to promote regulatory T cells, thereby reducing and Eliminate the toxic side effects of high doses that cause pulmonary edema.

Genes (named ABD/Fc/IL-2) encoding the above fusion proteins (fusion protein ABD/Fc/IL-2 (wild type) and fusion protein ABD/Fc/IL-2 (mutant type)) also belong to the present invention , gene is one of the following nucleotide sequences:

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

2) the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing;

3) The same as the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing A nucleotide sequence with more than 90% homology and high biological activity of the expressed protein;

4) Under high stringency conditions, it can be compared with the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID in the coding sequence listing. Nucleotide sequence to which the nucleotide sequence of NO: 13 hybridizes.

The high stringency condition is to wash the membrane at 65°C with a solution containing 0.1×SSPE (or 0.1×SSC) and 0.1% SDS after hybridization.

SEQ ID NO: 10 in the sequence listing is composed of 1314 bases, and its coding sequence is the 1-1314 bases from the 5' end, encoding a protein with the amino acid residue sequence shown in SEQ ID NO: 9 in the sequence listing , the 1-168 bases from the 5' end encode ABD, the 169-177 bases from the 5' end encode the connecting peptide, the 178-870 bases from the 5' end encode the human immunoglobulin Fc segment, Bases 871-915 from the 5' end encode the linker peptide, and bases 916-1314 from the 5' end encode IL-2. The nucleotide sequence encoding the amino acid residue sequence shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the Sequence Listing can easily be compared with those shown in SEQ ID NO: 9 according to the amino acid sequences of the three The residue differences in the amino acid sequence are determined by SEQ ID NO:10.

The polynucleotide encoding the fusion protein ABD/Fc/IL-2 of the present invention (including the fusion protein ABD/Fc/IL-2 (wild type) and the fusion protein ABD/Fc/IL-2 (mutant type)) may be in the form of DNA or RNA form. DNA forms include cDNA or synthetic DNA, which can be single-stranded or double-stranded, and can be either coding or non-coding.

Further, the variant of the polynucleotide encoding the fusion protein ABD/Fc/IL-2 of the present invention also belongs to the present invention, which encodes a polypeptide or polypeptide fragment having the same amino acid sequence as the fusion protein ABD/Fc/IL-2, Analogs and Derivatives. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants, and may also include substitutional, deletional, and insertional variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides that does not substantially alter the function of the encoded polypeptide .

The expression vector, transgenic cell line and host bacteria containing the gene (ABD/Fc/IL-2) of the present invention belong to the present invention.

Another object of the present invention is to provide a preparation method for recombinantly expressing the above fusion protein ABD/Fc/IL-2, which comprises the encoding gene (ABD/Fc/IL-2) containing the fusion protein ABD/Fc/IL-2 The recombinant expression vector is transformed or transduced into host cells, the host cells are cultured, and the protein is isolated and purified from the culture medium or cells to obtain the fusion protein ABD/Fc/IL-2.

In this method: the recombinant expression vector containing the gene encoding fusion protein ABD/Fc/IL-2 (ABD/Fc/IL-2) is to insert the gene encoding fusion protein ABD/Fc/IL-2 or its variant gene into into a recombinant expression vector. The starting vector for constructing the recombinant expression vector can be any bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other carriers. The starting vectors include, but are not limited to, expression vectors based on the T7 promoter for expression in bacteria, vectors for expression in mammalian cells, and vectors derived from baculovirus for expression in insect cells. In short, any plasmids and vectors can be used as long as they are replicable and stable in the host. An important feature of starting vectors is that they typically contain replication sites, promoters, marker genes and translational control elements.

Specifically, using the pcDNA3.1 vector as the starting vector, the constructed recombinant expression vector containing the gene encoding the fusion protein ABD/Fc/IL-2 was named pcDNA3.1/ABD/Fc/IL-2. The recombinant expression vector pcDNA3.1/ABD/Fc/IL-2 can be constructed using methods well known to those skilled in the art, such as in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombinant technology (Sambrook, et al Molecular cloing, a Laboratory Manual .Cold spring harbor laboratory. New York, 1989). The DNA sequence of the gene encoding the fusion protein ABD/Fc/IL-2 can be operably linked to an appropriate promoter in the expression vector to direct the synthesis of mRNA. The promoters can be: lac or trp promoters of E. coli, phage promoters, retroviruses and some other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.

In addition, the recombinant expression vector pcDNA3.1/ABD/Fc/IL-2 may also contain one or more selectable marker genes to provide a phenotypic shape for selection of transformed host cells, such as two for eukaryotic cell culture. Hydrofolate reductase gene, neomycin resistance gene and green fluorescent protein (GFP) gene or tetracycline or ampicillin resistance gene for Escherichia coli, etc. When the gene encoding the fusion protein ABD/Fc/IL-2 of the present invention is expressed in higher eukaryotic cells, in order to enhance transcription, it can also be inserted into the recombinant expression vector pcDNA3.1/ABD/Fc/IL-2 to enhance subsequence. Enhancers are cis-acting elements of DNA, usually 10-300 base pairs in length, that act on promoters to enhance transcription of genes. For example, the SV40 enhancer on the late side of the replication origin is about 100-270 base pairs in length, the polyoma enhancer or the adenovirus enhancer on the late side of the replication origin, etc.

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: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; eukaryotic cells such as yeast, plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS, 293 cells or Bowes melanoma cells .

Using conventional techniques well known to those skilled in the art, the recombinant expression vector pcDNA3.1/ABD/Fc/IL-2 can be transformed into host cells, the transformants are cultured, and the target protein (that is, fusion protein ABD/Fc/IL-2) can be induced to express, The target protein was isolated and purified.

Cultivate a host containing the encoding gene of the fusion protein ABD/Fc/IL-2 having both high biological activity and long plasma circulating half-life or high biological activity, long plasma circulating half-life and low toxic side effects of the present invention The culture medium and culture conditions of the cells can be both the culture medium and the culture conditions of the starting host.

Application of fusion protein ABD/Fc/IL-2 and its encoding gene to stimulate/activate high activity, long-acting (long in vivo circulating half-life) or both high activity, long-acting and low toxicity of immune cells of the body, the present invention also provides A drug for the treatment of various diseases, including malignant tumors, AIDS and viral hepatitis, has been developed. The active ingredient of the medicine comprises the above-mentioned fusion protein ABD/Fc/IL-2 or its encoding gene.

When the active pharmaceutical ingredient 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.

When necessary, one or more pharmaceutically acceptable carriers can also be added to the above-mentioned drugs. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants and adsorption carriers and the like conventional in the pharmaceutical field.

The medicament of the present invention can be made into various forms such as injection or freeze-dried powder. The medicines in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps 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).

The percentage concentration is mass/volume (W/V) percentage concentration or volume/volume (V/V) percentage concentration unless otherwise specified.

The DNA sequence synthesis and DNA sequence and amino acid sequence determination were all completed by Nanjing GenScript Biotechnology Company.

The ways of obtaining various biological materials described in the examples are only to provide an experimental way to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the prompts in the examples; All kinds of cells from mammals such as pigs or humans are in vitro, including those obtained from cell banks, or commercially purchased, and also prepared according to the introduction of existing literature, as well as those obtained from commercially available stem cells. induced by known methods.

The examples are implemented on the premise of the technical solutions of the present invention, and detailed implementations and specific operation procedures are given. The examples will help to understand the present invention, but the scope of the present invention is not limited to the following examples. .

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

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

ABD/Fc/IL-2 gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. according to CHO preference codon optimization, and the 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 the bases 1-1314 from the 5' end , encodes a protein with 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 from the 1-168th base of the 5' end, Bases 169-177 at the 5' end encode the connecting peptide, bases 178-870 at the 5' end encode the human immunoglobulin Fc segment, bases 871-915 at the 5' end encode the connecting peptide, and bases 871-915 at the 5' end encode the connecting peptide. The 916-1314th base of the 'end encodes IL-2.

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

Referring to the ExpiCHO-S ^TM operating instructions, the recombinant vector pcDNA3.1/ABD/Fc/IL-2 non-pyrogenic plasmid constructed in the above step 1.1 was transiently transfected into ExpiCHO-S ^TM cells at 37°C, 8% CO ₂ , Cultured at 120rpm; 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, identified by 12% SDS-PAGE electrophoresis and analyzed by Western blot.

The results are shown in Figure 1, in which panel A represents the identification result of SDS-PAGE electrophoresis of the purified fusion protein ABD-Fc-IL-2, panel B is the Western blot analysis of the results of panel A, in panels A and B , M represents Protein Maker; lane 1 represents reduction electrophoresis; lane 2 represents non-reduction electrophoresis. It was proved that the fusion protein ABD-Fc-IL-2 was obtained by expression and purification in this example, and it had high purity.

Example 2: Determination of specific protein activity of fusion protein ABD-Fc-IL-2

In this example, the specific protein activity of the fusion protein ABD-Fc-IL-2 obtained by expression and purification in Example 1 is determined with reference to the CTLL-2/MTT cell proliferation colorimetric method in the Chinese Pharmacopoeia (see, for example, patent document CN102372780A), which specifically includes the following step.

1) The national standard for the determination of the biological activity of recombinant human IL-2 (purchased from China National Institute for Food and Drug Control) 2000IU/mL was diluted 10 times to 200IU/mL according to the instructions, and the concentration of the assay was 0.1mg/mL. The fusion protein ABD-Fc-IL-2 was diluted to 1000-fold.

2) In a 96-well cell culture plate, make 2-fold serial dilutions of the recombinant IL-2 standard (200 IU/mL) and fusion protein ABD-Fc-IL-2 diluted in step 1), each with a total of 8 Dilution, make 2 duplicate wells for each dilution.

3) 50 μL of CTLL-2 cell (5×10 ⁵ /ml, ATCC TIB-214) suspension was added to each well, and cultured at 37° C. and 5% CO ₂ for 18-24 hours. Then, 20 μL of MTT solution was added to each well, and the cells were incubated at 37° C. and 5% CO ₂ for 4-6 hours.

4) Add 150 μL of cell lysate to each well and incubate at 37°C for 20 hours.

5) Measure the absorbance of the culture solution in each well at a wavelength of 570 nm, and record the measurement results.

6) Process with computer program or four-parameter regression calculation method. Calculate the half-effect dilution factor of the test product (ie fusion protein ABD-Fc-IL-2) (that is, the dilution factor from the test solution solution to the maximum effect point equivalent to 50% of the standard product), and calculate the supply according to the following formula 1. Trial price:

The titer of the test substance (IU/mL)=Pr×(Ds×Es)/(Dr×Er)(Formula 1)

Among them: Pr is the standard substance (here refers to the national standard for the determination of the biological activity of recombinant human IL-2) biological activity (titer), IU/mL; Ds is the test substance (here refers to the fusion protein ABD-Fc) -IL-2) pre-dilution ratio; Dr is the pre-dilution ratio of the standard product; Es is the dilution ratio of the test product equivalent to the half-effective dose of the standard product; Er is the dilution ratio of the half-effective dose of the standard product (EC ₅₀ ).

The results are shown in Figures 2 and 3, wherein Figure 2 shows the four-parameter regression fitting curve of recombinant human IL-2 standard (logarithm of Ig activity-OD ₅₇₀ value), and Figure 3 shows the fusion protein ABD/ Four-parameter regression fit curves of Fc/IL-2 and recombinant human IL-2 standards (Ig dilution factor - _OD570 value). Referring to the fitting curve formula in FIG. 2 and using the above formula 1, it can be calculated that the titer of the test fusion protein ABD-Fc-IL-2 is about 3.29×10 ⁷ IU/mL.

The specific activity of the test product (fusion protein ABD-Fc-IL-2) is calculated as: the ratio of the activity titer of the test product calculated by the above formula 1 to its protein concentration is the specific activity, and its unit is IU/mg . Therefore, it is calculated that the specific activity of the fusion protein ABD-Fc-IL-2 provided by the present invention is about 3.29×10 ⁸ IU/mg, which is higher than 1×10 ⁸ IU/mg, and according to the results shown in FIG. 3 , the fusion protein The specific activity of ABD-Fc-IL-2 was significantly higher than that of recombinant IL-2 standard (about 8.5×10 ⁶ IU/mg), and also significantly higher than that of the fusion protein IL-2 disclosed in documents 1 to 3 above. The specific activity of 2/HSA (up to 1.026×10 ⁷ IU·mg ⁻¹ ).

Example 3: Serum albumin binding assay of fusion protein ABD/Fc/IL-2

In this example, the Pull down/Western blot method is used to detect the binding of the fusion protein ABD/Fc/IL-2 expressed and purified in the above Example 1 to serum albumin, which specifically includes the following steps.

1) Experimental design:

Add 100ng ABD-Fc-IL-2 and 20μL Protein A gel to group A;

Add 100ng human serum albumin HSA and 20μL Protein A gel to group B;

In group C, 100 ng of ABD-Fc-IL-2, 100 ng of human serum albumin HSA and 20 μL of Protein A gel were added.

2) The mixtures of each group were rotated and incubated at 4°C for 4 hours, washed with PBST to remove unbound proteins, and detected by western blot.

The results are shown in FIG. 4 , it can be seen that the fusion protein ABD/Fc/IL-2 provided by the present invention can bind to human serum albumin (HSA) through the ABD in it, and then can make use of this binding property to make a protein with a longer circulating half-life in vivo. Serum albumin carries out macromolecular modification of the fusion protein, thereby prolonging the in vivo plasma circulation half-life of the fusion protein, and does not need to introduce human serum albumin with a larger molecular weight into the fusion protein, which is more conducive to the industrialized production of the fusion protein, And it is easier to penetrate tissue in vivo, so it is more suitable for clinical applications.

Example 4: Dynamic Detection of Fusion Protein ABD/Fc/IL-2 Plasma Concentration

In this example, the mouse pharmacokinetics of the fusion protein ABD/Fc/IL-2 obtained by expression and purification in the above Example 1 was tested to confirm that the fusion protein ABD/Fc/IL-2 indeed has a prolonged circulating half-life in vivo , which includes the following steps.

1) Select 5-6 week old BalB/c female mice, connect the full-length human immunoglobulin Fc segment and IL-2 with Fc/IL-2 (according to the same recombinant technology as in Example 1, and express it in CHO cells. The obtained fusion protein) was used as the control group, and the fusion protein ABD/Fc/IL-2 was used as the test group. In the control group and the test group, low (250 μg/kg, the dose of fusion protein, the same below) and medium (500 μg/kg) were set respectively. kg) and high (1000μg/kg) three dose groups were administered subcutaneously on the back of mice, and blood was collected from the tail at 30min, 1h, 2h, 4h, 8h, 12h, 24h, and 48h after injection, and serum was separated. Concentrations of Fc/IL-2 and fusion protein ABD/Fc/IL-2 in serum were detected over time.

2) The concentrations of fusion proteins ABD/Fc/IL-2 and Fc/IL-2 were detected by a double-antibody sandwich IL-2 ELISA kit, wherein the fusion proteins ABD/Fc/IL-2 and Fc/IL-2 were both measured at 250 , 125, 62.5, 31.25, 15.62, 7.81, 3.9pg/ml are diluted in equal proportions, and then do ELISA detection and draw a standard curve. The specific ELISA detection steps are as follows: (1) Set up two duplicate wells for the standard substance and the sample to be tested (serum sample), add 100 microliters, incubate at 37° C. for 90 minutes, and then wash the plate 4 times. (2) Add 100 μl of biotinylated antibody to each well, incubate at 37°C for 60 minutes, and wash the plate 4 times; (3) Add 100 μl of enzyme conjugate, incubate at 37°C for 30 minutes, and wash the plate 5 times; (4) ) Add 50 microliters of chromogenic substrate solution, incubate at 37°C for 15 minutes to terminate the reaction, and detect the OD ₄₅₀ value. Graphpad Prism 9 software was used for graphing and analysis, and Student's test was used for comparison between the two groups.

The changes in the concentration of fusion protein Fc/IL-2 and fusion protein ABD/Fc/IL-2 over time in serum are shown in Figure 5, where A, B and C represent low (250μg/kg) and medium (500μg, respectively) The pharmacokinetic curves of mice in three dose groups (1000μg/kg) and high (1000μg/kg) showed that in the dose range of 0.25-1mg/kg, the fusion proteins ABD-Fc-IL-2 and Fc-IL-2 were both It exhibits nonlinear pharmacokinetic characteristics, and is related to the pharmacokinetic characteristics of fusion protein Fc/IL-2 (the peak time of blood drug concentration for subcutaneous injection is about 2.5 hours, and no significant difference can be detected after about 20 hours of administration. Compared with the biological activity, the plasma circulating half-life is about 2.5 hours), the fusion protein ABD-Fc-IL-2 has a significantly longer action time in vivo (the peak time of blood drug concentration for subcutaneous injection is about 8 hours, and the time for administration is 48 hours). Significant biological activity can still be detected, the plasma circulating half-life is about 10 hours), and the clearance rate is lower. Therefore, the fusion protein ABD-Fc-IL-2 provided by the present invention can prolong the plasma circulating half-life of IL-2 in vivo, which is beneficial to ensure stable plasma concentration of IL-2.

The toxic and side effects of the existing IL-2 products and the fusion proteins provided in the above-mentioned documents 1 to 4 are mainly caused by the binding of IL-2 to α receptors and/or β receptors, so the present invention expresses the above-mentioned Example 1. After purification of the obtained fusion protein ABD-Fc-IL-2 (wild type), the native IL-2 was replaced by mutants of IL-2 (including, for example, IL-2v1, IL-2v2 and IL-2v3), and a A fusion protein with low toxicity and side effects, and the fusion protein has the same high biological activity, long plasma circulating half-life and pharmacokinetic characteristics as the fusion protein ABD-Fc-IL-2 obtained by expression and purification in the above Example 1, It is not repeated here.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Peking University

Sun Hongyan

<120> A fusion protein ABD/Fc/IL-2 and its encoding gene, preparation method and application

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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 Gln

65 70 75 80

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

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 Gln Pro

115 120 125

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

130 135 140

Lys Asn Gln 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 Gln 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 Gln Gln Gly Asn Val Phe

195 200 205

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

65 70 75 80

Asp Pro Arg Asp Val Val Ser Asn Ile 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 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> 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 Gln Gln 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 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Ala Pro Thr Ser 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 Phe Lys Phe Tyr 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 Leu 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> 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

aactccacct 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 agaacacagct 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 Gln Gln 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 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Ala Pro Thr Ser 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 Leu 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> 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 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 Gln Gln 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 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

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

290 295 300

Ser Ala Pro Thr Ser 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 (10)

1. A fusion protein ABD/Fc/IL-2, characterized in that the fusion protein ABD/Fc/IL-2 comprises human serum albumin binding domain ABD (comprising as shown in SEQ ID NO: 1 in the sequence listing) amino acid sequence), full-length human immunoglobulin Fc segment and IL-2 (comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing) or its mutants, and the fusion protein ABD/Fc/IL- 2 has high biological activity (protein specific activity is higher than 1×10 ⁸ IU/mg); The full-length human immunoglobulin Fc fragment is selected from human IgGl, IgG2, IgG3 and IgG4. 2 . The fusion protein ABD/Fc/IL-2 according to claim 1 , wherein the full-length human immunoglobulin Fc segment comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 3 . 3. The fusion protein ABD/Fc/IL-2 according to claim 1 or 2, wherein the mutant of IL-2 is at the 42nd, 45th, 72nd, Mutants obtained by mutating any one or more of positions 80, 81, 85, 86, and 92; Preferably, F42, Y45, L72, L80, R81, L85, I86, and I92 of the amino acid sequence of native IL-2 are mutated to A42, A45, A72, F80, D81, V85, V86, and F92, respectively; 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 native IL-2 F42 and Y45 of the amino acid sequence of IL-2v1 are mutated into A42 and A45 respectively; the mutant IL-2v2 is the mutant IL-2v1 whose amino acid sequence L72 is mutated to A72; the mutant IL-2v3 is the mutant IL-2v2 L80, R81, L85, I86, and I92 of the amino acid sequence were mutated to F80, D81, V85, V86, and F92, respectively; Further preferably, the mutant of IL-2 comprises the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in the sequence listing. 4. The fusion protein ABD/Fc/IL-2 according to any one of claims 1-3, characterized in that, the fusion protein ABD/Fc/IL-2 further comprises a connecting peptide or an analog thereof, using in linking the human serum albumin binding domain ABD, the full-length human immunoglobulin Fc segment and IL-2 or a mutant thereof into the fusion protein ABD/Fc/IL-2; Preferably, the linking peptide or its analog is used for linking the carboxy terminus of the human serum albumin binding domain ABD and the amino terminus of the Fc segment of a full-length human immunoglobulin, and for linking the full-length human immunoglobulin The carboxyl terminus of the protein Fc segment and the amino terminus of IL-2 or its mutants; Optionally, the amino acid residue sequence of the linking peptide is shown in SEQ ID NO: 7 or SEQ ID NO: 8 in the sequence listing. 5. The fusion protein ABD/Fc/IL-2 according to any one of claims 1-4, wherein the amino acid residue sequence of the fusion protein ABD/Fc/IL-2 is the following amino acid residues One of the base sequences: 1) the amino acid residue sequence shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing; 2) The amino acid residue sequence shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the sequence listing is subjected to substitution, deletion or addition of one to ten amino acid residues and the Amino acid sequence with high biological activity (specific protein activity higher than 1×10 ⁸ IU/mg). 6. The gene encoding fusion protein ABD/Fc/IL-2 (ABD/Fc/IL-2) according to any one of claims 1-5, the nucleotide sequence of which is one of the following nucleotide sequences : 1) the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing; 2) the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing; 3) The same as the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 in the coding sequence listing A nucleotide sequence with more than 90% homology and high biological activity (protein specific activity higher than 1×10 ⁸ IU/mg) after the expressed protein; 4) Under high stringency conditions, it can be compared with the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID in the coding sequence listing. Nucleotide sequence to which the nucleotide sequence of NO: 13 hybridizes. 7. A recombinant expression vector pcDNA3.1-ABD/Fc/IL-2, wherein the recombinant expression vector pcDNA3.1-ABD/Fc/IL-2 comprises the encoding gene (ABD) of claim 6. /Fc/IL-2) for expressing the fusion protein ABD/Fc/IL-2 according to any one of claims 1-5. 8. A transgenic cell line or host bacteria, characterized in that, the encoding gene (ABD/Fc/IL-2) according to claim 6 or the recombination according to claim 7 are included in the transgenic cell line or host bacteria The expression vector pcDNA3.1-ABD/Fc/IL-2 is used to express the fusion protein ABD/Fc/IL-2 according to any one of claims 1-5. 9. a kind of medicine, it is used for the treatment of malignant tumor, infectious disease and body immunity is low, and described medicine comprises fusion protein ABD/Fc/IL-2 described in any one of claim 1-5 or claim 6. The encoding gene (ABD/Fc/IL-2) is used as an active ingredient; Optionally, the medicament further comprises one or more of the following: pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, Surfactant. 10. A method for recombinantly expressing the fusion protein ABD/Fc/IL-2 according to any one of claims 1-5, comprising the steps of: 1) construct the described transgenic cell line or host bacterium of claim 8, and cultivate this transgenic cell line or host bacterium; 2) separating and purifying the protein from the culture medium or cell of the transgenic cell line or host bacteria to obtain the fusion protein ABD/Fc/IL-2; Preferably, the method specifically includes the following steps: a) construct a recombinant expression vector pcDNA3.1-ABD/Fc/IL-2 containing the encoding gene (ABD/Fc/IL-2) of claim 6; b) transforming the recombinant expression vector pcDNA3.1-ABD/Fc/IL-2 into a transgenic cell line or host strain, and culturing and expressing to obtain the fusion protein ABD/Fc/IL-2; c) Purify the fusion protein ABD/Fc/IL-2 by affinity chromatography or ion exchange chromatography.

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