WO2021043127A1 - Chimeric protein - Google Patents

Abstract

Disclosed is a new chimeric protein, comprising a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises a coagulation factor and a first Fc variant of an Fc domain of an immunoglobulin, and the second polypeptide chain comprises a second Fc variant of the Fc domain of the immunoglobulin. The first Fc variant and the second Fc variant comprise an FcRn-binding site. The chimeric protein has a coagulation factor activity and a prolonged half-life, and can be used in the treatment of bleeding diseases such as hemophilia.

Description

Chimeric protein Technical field

The present invention relates to the field of biomedicine. More specifically, the present invention relates to a therapeutic chimeric protein composed of two polypeptide chains.

Background technique

Coagulation factors are various protein components involved in the blood clotting process. Its physiological function is to be activated when blood vessels bleed, stick to platelets and plug the leaks in blood vessels. The types of coagulation factors include coagulation factors Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅶ, Ⅷ, Ⅸ, Ⅹ, Ⅺ, Ⅻ, XIII, etc., which all play an important role in the coagulation process. Among them, coagulation factors Ⅶ, Ⅷ and Ⅸ have been made into pharmaceutical preparations for the treatment of bleeding diseases, especially for hemophilia.

Hemophilia B (congenital coagulation factor IX deficiency, or Christmas disease) is a recessive inherited bleeding disorder linked to the X chromosome. It is caused by mutations in the patient’s coagulation factor IX (FIX) gene. Caused by deficiency, it is one of the common types of hemophilia. It leads to reduced coagulation activity in vivo and in vitro and requires lifelong medical monitoring of diseased individuals. In the absence of intervention, spontaneous bleeding in the joints of the affected individual will cause severe pain and make the person weak and unable to move; if bleeding into the muscles, it will cause blood to accumulate in such tissues; throat and neck Internal spontaneous bleeding, if not treated in time, can cause asphyxiation; in addition, severe bleeding after renal bleeding, surgery, minor accidental injury, or tooth extraction is also very common. Hemophilia B accounts for 15%-20% of all hemophilia patients. The number of patients with hemophilia B in China is about 5,300-7,100.

Coagulation factor IX (FIX) is used to control and prevent bleeding in patients with hemophilia B, including bleeding control and prevention during surgery.

There are currently three generations of drugs for the treatment of hemophilia B that have been marketed. The first generation is the prothrombin complex, which is a plasma source, and has the problems of difficulty in purification, short half-life, and frequent administration. Its half-life is only 18-24h, and because it is a plasma source, there are some problems that have spread. The potential risks of known or unknown pathogens should be weighed in clinical use; the second generation is the conventional half-life recombinant coagulation factor IX, which represents the product BeneFIX, which is expressed in animal cells. It also has the problem of short half-life, with a half-life of 18-24h and relative For the first generation product, because it has a lower incremental recovery (K value), a higher dose than the first generation product must be used; the third generation is long-acting recombinant coagulation factor IX, which is derived from animal cells Expressed, the representative products are Alprolix, Idelvion and Rebinyn (N9-GP), which have a half-life extension of 3-5 times compared to the first and second generation products. At present, there are no second-generation and third-generation products on the market in China.

Although the third-generation product has modified the coagulation factor IX to extend the half-life of the coagulation factor IX, for example, Alprolix is the expression of recombinant coagulation factor IX (rFIX) and natural Fc, and Idelvion is the combination of recombinant coagulation factor IX (rFIX) and white coagulation. Protein (albumin) fusion expression, Rebinyn is the PEGylation of recombinant coagulation factor IX (rFIX). However, the above-mentioned related products still have the following problems: 1) Due to the low rate of correct pairing of the two asymmetric polypeptide chains of the product, the product yield is low, the purity is low, and the purification difficulty and purification cost are high in large-scale production; 2) The half-life needs to be further extended.

Therefore, in the field of hemophilia treatment, there is an urgent need to provide a compound that can be obtained with higher yield and purity, and at the same time has a comparable or longer half-life compared with the existing third-generation products.

Summary of the invention

The first aspect of the present invention provides a new chimeric protein, which can be obtained with a higher yield and purity, has a higher yield, and is compatible with the existing third-generation product (long-acting recombinant coagulation factor). IX) Compared with that, it has a comparable or longer half-life.

In the first aspect, the present invention provides a new chimeric protein comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a coagulation factor and an immunoglobulin Fc structure Domain of the first Fc variant, the second polypeptide chain comprising the second Fc variant of the Fc domain of the immunoglobulin, the first Fc variant and the second Fc variant comprising FcRn binding Site, wherein the first Fc variant contains the following amino acid mutations: from the N-terminus of the Fc domain of the immunoglobulin, the amino acid at position 146 is serine, and the amino acid at position 148 is alanine , And the amino acid at position 187 is valine, and the second Fc variant contains the following amino acid mutation: from the N-terminus of the Fc domain of the immunoglobulin, the amino acid at position 146 is tryptophan. Through a large number of experiments, the inventors surprisingly discovered that the combination of the site-directed mutation of the first Fc variant and the site-directed mutation of the second Fc variant is compared with the situation where the Fc domain of immunoglobulin is not mutated (for example, Alprolix), or other site-directed mutagenesis, on the one hand, it can significantly reduce the mismatch rate of two asymmetric polypeptide chains, significantly improve the purity and yield of the resulting chimeric protein, and on the other hand, it can significantly improve the The output of the chimeric protein can meet the needs of large-scale production, reduce production costs, reduce the difficulty of purification during large-scale production, and improve production efficiency. For example, when the amino acid mutation of the first Fc variant is interchanged with the amino acid mutation of the second Fc variant, it will result in a significant reduction in the pairing accuracy of the two polypeptide chains obtained, and the resulting chimera Significant reduction in protein yield.

Optionally, the immunoglobulin is IgG, preferably, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4; more preferably, the immunoglobulin is human IgG1, IgG2, IgG3, or IgG4; further preferably, The immunoglobulin is human IgG1 or IgG2. More preferably, the immunoglobulin of the first polypeptide chain and the second polypeptide chain are the same, and more preferably the first polypeptide chain and the second polypeptide chain The immunoglobulin is human IgG1.

Optionally, the sequence of the first Fc variant is shown in SEQ ID NO. 1 and the sequence of the second Fc variant is shown in SEQ ID NO. 2.

Optionally, the coagulation factor is coagulation factor IX, coagulation factor VIII, or coagulation factor VII, preferably coagulation factor IX, preferably the coagulation factor is human coagulation factor IX, and more preferably the sequence of the coagulation factor is as SEQ ID NO. 25 Shown.

Optionally, the sequence of the first polypeptide chain is shown in SEQ ID NO. 3 and the sequence of the second polypeptide chain is shown in SEQ ID NO. 4.

Further preferably, the first Fc variant and the second Fc variant further comprise the following amino acid mutations: from the N-terminus of the Fc domain of the immunoglobulin, the amino acid at position 32 is tyrosine , The amino acid at position 34 is threonine, and the amino acid at position 36 is glutamic acid. The inventors found that by making the first Fc variant and the second Fc variant further contain the three amino acid mutations simultaneously, it is possible to obtain a longer half-life than Alprolix and a reduction in the first polypeptide chain. A chimeric protein with a mismatch rate with the second polypeptide chain. The chimeric protein can be obtained in a higher yield, and the coagulation activity of the chimeric protein is basically equivalent to that of Alprolix and Benefix, indicating that the chimeric protein of the present invention It is expected to provide a better medication option for patients with hemophilia B.

Optionally, the sequence of the first Fc variant is shown in SEQ ID NO. 5, and the sequence of the second Fc variant is shown in SEQ ID NO. 6.

Optionally, the sequence of the first polypeptide chain is shown in SEQ ID NO. 7, and the sequence of the second polypeptide chain is shown in SEQ ID NO. 8.

The second aspect of the present invention provides a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule comprising a first nucleotide sequence encoding the first polypeptide chain of the chimeric protein of the first aspect of the present invention, and The second nucleic acid molecule comprises a second nucleotide sequence encoding the second polypeptide chain of the chimeric protein of the first aspect of the invention.

Optionally, the first nucleotide sequence is shown in SEQ ID NO. 9, and further preferably, the first nucleotide sequence is shown in SEQ ID NO. 11.

Optionally, the second nucleotide sequence is shown in SEQ ID NO.10, and further preferably, the second nucleotide sequence is shown in SEQ ID NO.12.

The third aspect of the present invention provides an expression vector for expressing the chimeric protein according to the first aspect of the present invention. The expression vector comprises the first nucleic acid molecule and the second nucleic acid molecule according to the second aspect of the present invention. The nucleic acid molecule comprises the first nucleotide sequence encoding the first polypeptide chain of the chimeric protein according to the first aspect of the present invention, and the second nucleic acid molecule comprises the second polynucleotide encoding the chimeric protein according to the first aspect of the present invention. The second nucleotide sequence of the peptide chain.

Optionally, the first nucleotide sequence is shown in SEQ ID NO. 9, and the second nucleotide sequence is shown in SEQ ID NO. 10.

Optionally, the first nucleotide sequence is shown in SEQ ID NO. 11, and the second nucleotide sequence is shown in SEQ ID NO. 12.

Optionally, the expression vector includes two promoters, and the promoters may be, for example, a CMV promoter and/or an EF1α promoter. Preferably, the expression vector contains two identical promoters, and more preferably, the two identical promoters are CMV promoters. The inventor unexpectedly discovered that when the expression vector contains two identical promoters, preferably two CMV promoters, compared to the expression vector containing two different promoters, for example, it contains a CMV promoter and an EF1α promoter. It can significantly reduce the mismatch rate between the first polypeptide chain and the second polypeptide chain, thereby significantly improving the yield and/or purity of the chimeric protein.

Optionally, the first nucleotide sequence is upstream of the second nucleotide sequence. The inventors unexpectedly discovered that when the first nucleotide sequence is upstream of the second nucleotide sequence, the mismatch rate of the expressed chimeric protein is compared with that of the first nucleotide sequence in the second nucleotide sequence. When the acid sequence is downstream, there is a significant decrease, and the yield of the resulting chimeric protein is greatly improved compared to when the first nucleotide sequence is downstream of the second nucleotide sequence.

The fourth aspect of the present invention also provides a host cell, the host cell comprising the expression vector of the third aspect of the present invention.

Optionally, the host cell is a mammalian cell, preferably, the host cell is a CHO cell.

The fifth aspect of the present invention also provides a pharmaceutical composition, which comprises the chimeric protein according to the first aspect of the present invention and pharmaceutically acceptable excipients.

The sixth aspect of the present invention also provides the use of the chimeric protein of the first aspect of the present invention in the preparation of a medicament for treating patients suffering from diseases that benefit from the administration of coagulation factors.

Optionally, the disease is selected from coagulation disorders, bleeding disorders, hemophilia or bleeding disorders.

Optionally, the hemophilia is hemophilia B or hemophilia A. Preferably, the hemophilia is hemophilia B.

Description of the drawings

Figure 1: Schematic diagram of the structure of the chimeric protein in one embodiment of the present invention.

Figure 2: Example 1-2 and Comparative Example 1-6 in the process of preparing the chimeric protein, the coagulation activity detection results of the cell fermentation broth supernatant, where the ordinate "Activity" represents the coagulation activity.

Figure 3: The blood coagulation activity test results of the cell fermentation broth supernatant when different expression vectors are used in Comparative Examples 7-9 and Example 2, where the ordinate represents the blood coagulation activity.

Figure 4: SDS-PAGE detection results of the expressed chimeric protein when different expression vectors are used in Comparative Examples 7-9 and Example 2.

Figure 5: Half-life of ALPROLIX and the title compound of Example 2 in FIX-deficient coagulation dysfunction mice.

detailed description

Figure 1 schematically shows the structure of a chimeric protein (FIX-Fc1: Fc2) in an embodiment of the present invention. The chimeric protein comprises a first polypeptide chain and a second polypeptide chain. The polypeptide chain includes the coagulation factor IX and the first Fc variant of the Fc domain of immunoglobulin. In FIG. 1, the first polypeptide chain is schematically shown as "FIX-Fc1", and FIX stands for coagulation factor. IX, Fc1 represents the first Fc variant in the first polypeptide chain; the second polypeptide chain includes the second Fc variant of the Fc domain of the immunoglobulin, in Figure 1, the second The polypeptide chain is schematically shown as "Fc2", where "Fc2" means the second Fc variant in the second polypeptide chain. Both the first Fc variant and the second Fc variant comprise an FcRn binding site. In humans, the Fc domain of immunoglobulins can bind to the receptor FcRn, thereby prolonging the half-life of FIX.

In one embodiment of the present invention, the immunoglobulin is human immunoglobulin G1 (IgG1). In humans, the Fc domain of human immunoglobulin G1 (IgG1) can bind to the receptor FcRn, thereby prolonging the half-life of FIX linked to it in humans (such as the third-generation Alprolix). In one embodiment of the present invention, by site-directed mutagenesis of the Fc domain of human immunoglobulin G1 (IgG1), the resulting chimeric protein can further extend the half-life of FIX in the human body and reduce the chimeric protein compared to Alprolix. The mismatch rate during the expression of the synthin improves the yield, purity, and yield of the obtained chimeric protein.

definition

Immunoglobulin

The chimeric protein of the invention comprises at least a part of an immunoglobulin constant region. Immunoglobulins are composed of four protein chains covalently associated-two heavy chains and two light chains. Each chain further consists of a variable region and a constant region. Depending on the isotype of the immunoglobulin, the heavy chain constant region consists of 3 or 4 constant region domains (e.g. CH1, CH2, CH3, CH4). Certain isotypes also contain hinge regions.

The term "Fc region" or "Fc domain" as used herein is defined as the part of the constant region of the heavy chain. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the Fc region is usually defined as starting at the papain cleavage site Click the upstream hinge region and terminate at the C-terminus of the antibody. Correspondingly, the complete Fc region includes at least a hinge domain, a CH2 domain, and a CH3 domain. The "Fc domain of immunoglobulin" herein refers to the natural Fc domain of immunoglobulin, which is defined as the part of immunoglobulins numbered 221-247 according to the EU numbering system. Said immunoglobulin especially refers to humans. Immunoglobulin, in particular, refers to human immunoglobulin G, such as human immunoglobulin G1, human immunoglobulin G2, human immunoglobulin G3 or human immunoglobulin G4. The numbering of the amino acid mutation sites contained in the Fc variant of the chimeric protein of the present invention is calculated starting from the first amino acid at the N-terminus of the Fc domain of the immunoglobulin.

As used herein, "EU numbering system" or "EU index" is usually used when referring to residues in the constant region of an immunoglobulin heavy chain (for example, Kabat et al., Sequences of Immunological Interest, Fifth Edition, Public Health Service The EU index reported in National Institutes of Health, Bethesda, Md. (1991); the hinge region in the constant region of the heavy chain is approximately residues 216-230 (EU numbering) of the heavy chain). For example, "EU index in Kabat" refers to the residue number of human IgG1EU antibody. For residues in the constant region of immunoglobulins, see Figures 40A-40D in U.S. Provisional Application No. 60/640,323 for examples of numbering according to the EU numbering system.

The term "native Fc" or "native Fc domain" or "Fc domain" as used herein refers to a molecule containing a sequence of non-antigen-binding fragments resulting from or otherwise produced by antibody digestion, whether in monomeric or multimeric form , And can include a hinge area. The immunoglobulin source of natural Fc is preferably human and the immunoglobulin can be any immunoglobulin, preferably, the immunoglobulin is IgG1 or IgG2, more preferably, the immunoglobulin is human immunoglobulin G1 and Human immunoglobulin G2. Natural Fc molecules are composed of monomeric polypeptides, or dimers or multimers that are covalently (ie, disulfide bonds) and non-covalently bound. The number of intermolecular disulfide bonds between monomer subunits of natural Fc molecules ranges from 1 to 4, depending on the type (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl and IgGA2). An example of natural Fc is the disulfide-bonded dimer produced by papain digestion of IgG. Another example of natural Fc is the part of human immunoglobulin IgG1 kabat numbered 221-447. The term "native Fc" as used herein includes, but is not limited to, monomer, dimer, and multimer forms.

The term "Fc variant" or "Fc domain variant" as used herein refers to a molecule that is derived from natural Fc or natural Fc domain through amino acid modification but still contains a binding site for binding to the receptor FcRn (nascent Fc receptor) Or sequence. The term "Fc variant" or "Fc domain variant" also includes molecules or sequences derived from humanization of non-human natural Fc. In addition, the term "Fc variant" or "Fc domain variant" also includes molecules or sequences that lack one or more native Fc sites or residues, or where one or more Fc sites or residues have been The modified molecule or sequence, the Fc site or residue affects or participates in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) when in the selected host cell N-terminal heterogeneity during expression, (4) glycosylation, (5) interaction with complement, or (6) antibody-dependent cytotoxicity (ADCC).

A coagulation factor, as used herein, refers to any naturally or recombinantly produced molecule or analog thereof that prevents or shortens the duration of bleeding episodes in a subject suffering from a hemostatic disorder. In other words, clotting factor refers to any molecule that has clotting activity.

Polypeptide is used herein to refer to an amino acid polymer, but does not refer to a product of a specific length; therefore, peptides, oligopeptides, and proteins are all included in the definition of polypeptide. This term does not exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, pegylation, the addition of lipid moieties, or the addition of any organic or inorganic molecules. Included in this definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and polypeptides containing substituents, as well as other naturally or non-naturally modified polypeptides known in the art.

As used herein, "Factor IX" or "FIX" polypeptide refers to any Factor IX polypeptide, including but not limited to recombinantly produced polypeptides, synthetically produced polypeptides, and Factor IX polypeptides extracted or isolated from cells or tissues, the cells and Tissues include but are not limited to liver and blood. Other names that can be used interchangeably with factor IX include factor 9, Christmas factor, plasma coagulation kinase (PTC), coagulation factor IX, and serum factor IX. Abbreviations for factor IX include FIX and F9. Factor IX includes related polypeptides from different species, including but not limited to animals of human and non-human origin. Human factor IX (hFIX) includes factor IX, allelic variant isoforms or mutated allelic variants, molecules synthesized from nucleic acids, proteins isolated from human tissues and cells, and modified forms thereof. The FIX polypeptides provided herein can be further modified, such as by chemical modification or post-translational modification. Such modifications include, but are not limited to, glycosylation, pegylation, albuminization, farnysylation, carboxylation, hydroxylation, phosphorylation, and other peptide modifications known in the art.

Factor IX includes factor IX from any species, including human and non-human species. FIX polypeptides of non-human origin include, but are not limited to, murine, dog, cat, rabbit, bird, cow, sheep, pig, horse, fish, frog, and other primate factor IX polypeptides.

FIX polypeptides also include precursor polypeptides and mature FIX polypeptides in single-chain or double-chain forms, truncated forms that have activity, and include allelic variants and species variants, gene-encoded splicing variants, and other variants, Also includes modified FIX polypeptides. It also includes polypeptides that retain at least FIX activity, such as FVIIIa binding activity, factor X binding activity, phospholipid binding activity, and/or coagulant activity of the polypeptide. For retained activity, the activity can be altered, such as reduced or increased compared to wild-type FIX, as long as the level of retained activity is sufficient to produce a detectable effect. FIX polypeptides include, but are not limited to, tissue-specific isotypes and allelic variants, synthetic molecules prepared by translation of nucleic acid molecules, proteins generated by chemical synthesis, such as the synthesis of linked shorter polypeptides, and those produced by recombinant methods. Proteins, proteins isolated from human and non-human tissues and cells, chimeric FIX polypeptides and their modified forms. FIX polypeptides also include fragments or portions of FIX that are of sufficient length or include an appropriate region to retain (if necessary, when activated) at least one activity of the full-length mature polypeptide. FIX polypeptides also include those that contain chemical or post-translational modifications and those that do not contain chemical or post-translational modifications. Such modifications include, but are not limited to, pegylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

As used herein, the "activity" of a FIX polypeptide refers to any activity exhibited by the Factor IX polypeptide. Such activities can be tested in vitro and/or in vivo, including but not limited to coagulation or coagulant activity, procoagulant activity, proteolytic or catalytic activity such as achieving factor X (FX) activation; antigenicity (binding to anti-FIX The ability of an antibody or the ability to compete with a polypeptide for binding to an anti-FIX antibody); the ability to bind factor VIIIa or factor X; and/or the ability to bind to phospholipids. The activity can be assessed in vitro or in vivo by using known assays, for example by measuring coagulation in vitro or in vivo. The results of such assays indicate that the polypeptide exhibits an activity that can be correlated with the in vivo activity of the polypeptide, where in vivo activity can refer to biological activity. Assays for determining the functionality or activity of modified forms of FIX are known to those skilled in the art. Exemplary assays for assessing the activity of FIX polypeptides include prothrombin prokinase time (PT) assays or activated partial thromboplastin time (aPTT) assays to assess coagulant activity, or the use of synthetic substrates to assess catalytic or proteolytic activity Determination of color development.

As used herein, "nucleic acid molecule" or "nucleic acid" includes DNA, RNA and analogs thereof, including peptide nucleic acid (PNA) and mixtures thereof. Nucleic acids can be single-stranded or double-stranded. The nucleic acid molecule encoding the chimeric protein of the present invention provided by the present invention includes any allelic variant or splice variant of the encoded chimeric protein.

As used herein, a vector refers to a discrete element used to introduce exogenous nucleic acid into a cell for its expression or replication. The vector usually exists in the form of a plasmid, but it can also be designed to integrate the gene or part of it into the chromosome of the genome. It also covers carriers as artificial chromosomes, such as bacterial artificial chromosomes, yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

As used herein, an expression vector includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region capable of achieving expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain both elements. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into a suitable host cell, will lead to the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those replicable in eukaryotic and/or prokaryotic cells, as well as those in the form of plasmids or those integrated into the host cell genome.

As used herein, "yield" or "yield of chimeric protein" is expressed by the coagulation activity per milliliter of cell fermentation supernatant of cells expressing the chimeric protein, and the unit is IU/ml. Among them, IU is the abbreviation of International Unit.

Example

The following examples further illustrate the present invention. It should be noted that these embodiments do not constitute a limitation on the protection scope of the present invention.

Example 1 Preparation of Chimeric Protein 1

Chimeric protein 1: The schematic diagram of the structure of chimeric protein 1 is shown in Figure 1, which includes a first polypeptide chain (schematically represented by "FIX-Fc1" in Figure 1) and a second polypeptide chain (shown in Figure 1 The “Fc2” of the Fc2 is schematically indicated), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.3, and the first Fc variant (such as the Fc domain of coagulation factor IX and human immunoglobulin G1) is shown in SEQ ID NO. SEQ ID NO. 1), the second polypeptide chain (as shown in SEQ ID NO. 4) comprises a second Fc variant of the Fc domain of human immunoglobulin G1 (as shown in SEQ ID NO. 2) Show). SEQ ID NO.1 contains the following 3 amino acid mutations: from the N-terminus of the Fc domain of human immunoglobulin G1, the 146th amino acid is serine, the 148th amino acid is alanine, and the 187th The amino acid at position is valine. SEQ ID NO. 2 contains the following amino acid mutation: from the N-terminus of the Fc domain of human immunoglobulin G1, the 146th amino acid is tryptophan.

1) Construction of expression vector 1

Take pOptiVEC ^TM-

The vector (purchased from Gibco) was used as a template, PCR amplified CMV promoter fragments, and restriction sites (NheI and NotI) were added at the beginning and the end, and then ligated to pBudCE4.1 vector (purchased from Gibco) to replace the original Some EF1α promoters are used to obtain the pBudCE4.1R vector.

According to the nucleotide sequence shown in SEQ ID NO.9, the first nucleotide sequence of the first polypeptide chain of the whole gene synthesis and expression chimeric protein 1, and the nucleotide sequence shown in SEQ ID NO.10 , Synthesize the second nucleotide sequence of the second polypeptide chain expressing chimeric protein 1.

The nucleotide sequence shown in SEQ ID NO. 9 obtained in the previous step is added with restriction sites (XhoI and BamHI) at the beginning and end, and inserted into the downstream expression region of the first CMV promoter upstream of the pBudCE4.1R vector. The nucleotide sequence shown in SEQ ID NO. 10 obtained in the previous step is added with restriction sites (NotI and MluI) at the beginning and end, and inserted into the downstream expression region of the second CMV promoter downstream of the pBudCE4.1R vector. The FIX-pBudCE4.1R-1 vector (referred to as "expression vector 1") is obtained.

2) Expression of chimeric protein 1

The expression vector 1 was transiently transfected into 293F cells (purchased from Gibco), the transfected 293F cells were cultured, the 293F cell fermentation broth was collected, centrifuged, and filtered to obtain the chimeric protein 1.

In the following example 2, the chimeric protein 2-7 synthesized in the control examples 1-6 and the chimeric protein in Alprolix have similar structures to the chimeric protein 1. The schematic diagrams of the structure are shown in Figure 1, and also include the first poly A peptide chain (schematically represented by "FIX-Fc1" in Figure 1) and a second polypeptide chain (schematically represented by "Fc2" in Figure 1), the first polypeptide chain comprising coagulation factor IX and human The first Fc variant of the Fc domain of immunoglobulin G1, the second polypeptide chain comprising the second Fc variant of the Fc domain of human immunoglobulin G1, the difference is that in the chimeric protein 2-7 The first Fc variant ("Fc1") and the second Fc variant ("Fc2") contain amino acid mutations that are different from the first Fc variant and the second Fc variant in chimeric protein 1. Specifically, Table 1 shows the amino acid mutations contained in the first Fc variant and the second Fc variant of the chimeric protein 1-7.

Table 1 Amino acid mutations contained in chimeric proteins 1-7

Note: The mutant amino acids in Table 1 are numbered from the N-terminus of the Fc domain of human immunoglobulin G1

Example 2 Preparation of Chimeric Protein 2

1) Construction of expression vector 2

Through the similar steps as in Example 1, the first nucleotide sequence of the first polypeptide chain expressing chimeric protein 2 (shown in SEQ ID NO.11) and the second polypeptide expressing chimeric protein 2 were synthesized The second nucleotide sequence of the chain (as shown in SEQ ID NO. 12).

The pBudCE4.1R vector containing two CMV promoters was obtained through the same steps as in Example 1. The first nucleotide sequence shown in SEQ ID NO. 11 is obtained, and restriction sites are added at the beginning and end, and inserted into the downstream expression region of the first CMV promoter upstream of the pBudCE4.1R vector; SEQ ID NO. will be obtained. In the second nucleotide sequence shown in 12, restriction sites are added at the beginning and end, and inserted into the downstream expression region of the second CMV promoter downstream of the vector. The FIX-pBudCE4.1R-2 vector (referred to as "expression vector 2") is obtained.

2) Expression of Chimeric Protein 2

Using a method similar to step 2) of Example 1, chimeric protein 2 was obtained.

Comparative Example 1 Preparation of Chimeric Protein 3

The chimeric protein 3 was obtained through the steps similar to those in Examples 1 and 2. The difference is that the first nucleotide sequence of the first polypeptide chain expressing the chimeric protein 3 is shown in SEQ ID NO. 13, which expresses the chimeric protein. The second nucleotide sequence of the second polypeptide chain of protein 3 is shown in SEQ ID NO.14.

Comparative Example 2 Preparation of Chimeric Protein 4

The chimeric protein 4 was obtained through the steps similar to those in Examples 1 and 2. The difference is that the first nucleotide sequence of the first polypeptide chain expressing the chimeric protein 4 is shown in SEQ ID NO. 15, which expresses the chimeric protein. The second nucleotide sequence of the second polypeptide chain of protein 4 is shown in SEQ ID NO.16.

Comparative Example 3. Preparation of Chimeric Protein 5

The chimeric protein 5 was obtained through the steps similar to those in Examples 1 and 2. The difference is that the first nucleotide sequence of the first polypeptide chain of the chimeric protein 5 is shown in SEQ ID NO. 17, and the chimeric protein is expressed. The second nucleotide sequence of the second polypeptide chain of protein 5 is shown in SEQ ID NO.18.

Comparative Example 4. Preparation of Chimeric Protein 6

The chimeric protein 6 was obtained through the steps similar to those in Examples 1 and 2. The difference is that the first nucleotide sequence of the first polypeptide chain of the chimeric protein 6 is shown in SEQ ID NO. 19, and the chimeric protein 6 is expressed. The second nucleotide sequence of the second polypeptide chain of protein 6 is shown in SEQ ID NO.20.

Comparative Example 5. Preparation of Chimeric Protein 7

The chimeric protein 7 was obtained through the steps similar to those in Examples 1 and 2. The difference is that the first nucleotide sequence of the first polypeptide chain of the chimeric protein 7 is shown in SEQ ID NO. 21, and the chimeric protein 7 is expressed. The second nucleotide sequence of the second polypeptide chain of protein 7 is shown in SEQ ID NO.22.

Comparative Example 6. Preparation of chimeric protein in Alprolix

The chimeric protein in Alprolix was obtained through the steps similar to those in Examples 1 and 2, except that the first nucleotide sequence of the first polypeptide chain expressing the chimeric protein in Alprolix is shown in SEQ ID NO.23 The second nucleotide sequence of the second polypeptide chain expressing the chimeric protein in Alprolix is shown in SEQ ID NO.24.

Example 3 Yield and mismatch rate detection

Yield detection

Under the same other conditions, use the coagulation factor kit (purchased from HYPHEN BioMed) to detect the activity of the 293F cell fermentation supernatant collected in the process of preparing the chimeric protein in Example 1-2 and Control Example 1-6. Blood coagulation activity, the results are shown in Figure 2: Chimeric protein 1-2 group has the highest coagulation activity, that is, the highest yield.

Mismatch rate detection

Under the same other conditions, the supernatant of the 293F cell fermentation broth collected during the preparation of the chimeric protein in Example 1-2 and Comparative Example 1-6 was purified by ProteinA (purchased from GE), and then SDS-PAGE The test results are shown in Table 2: Chimeric protein 1-2 groups have the highest purity or yield, the homologous single-stranded mismatch rate is the lowest, and the mismatch rate is much lower than that of Alprolix's chimeric protein.

Table 2 Test results of mismatch rate

Comparative Example 7 Construction of expression vector 8

The expression vector 8 was obtained through the similar steps as in Example 2. The difference is that when constructing the expression vector 8, the second nucleotide sequence (SEQ ID NO. 12) of Example 2 was added with restriction sites at the beginning and the end. Insert the downstream expression region of the first CMV promoter upstream of the pBudCE4.1R vector; insert the first nucleotide sequence (SEQ ID NO.11) of Example 2 with restriction sites at the beginning and end, and insert it into the downstream of the pBudCE4.1R vector The downstream expression region of the second CMV promoter.

Comparative Example 8 Construction of expression vector 9

The expression vector 9 was constructed using similar steps as in Example 2, except that the second nucleotide sequence (SEQ ID NO. 12) of Example 2 was inserted into the CMV promoter of the pBudCE4.1 vector (purchased from Gibco) Downstream expression region: The first nucleotide sequence (SEQ ID NO.11) of Example 2 was added with restriction sites at the beginning and end, and inserted into the EF1α promoter region downstream of the pBudCE4.1 vector to obtain expression vector 9.

Comparative Example 9 Construction of expression vector 10

The expression vector 10 was constructed using similar steps as in Example 2, except that the first nucleotide sequence (SEQ ID NO.11) of Example 2 was inserted into the CMV starter of the pBudCE4.1 vector (purchased from Gibco) The second nucleotide sequence (SEQ ID NO.12) of Example 2 was added with restriction sites at the beginning and end, and inserted into the EF1α promoter region downstream of the pBudCE4.1 vector to obtain the expression vector 10.

Example 4 Comparison of the yield and mismatch rate of chimeric proteins expressed by expression vectors 2, 8-10

Yield detection

The vectors obtained in Comparative Examples 7-9 were respectively transiently transfected into 293F cells, the transfected 293F cells were cultured, the 293F cell fermentation broth was collected, and the chimeric protein was harvested by centrifugation and filtration. Under the same other conditions, the coagulation activity of the 293F cell fermentation supernatant collected in the process of preparing the chimeric protein in Example 2 and Comparative Example 7-9 was tested by the coagulation factor kit activity, and the results are shown in Figure 3: The expression vector 10 and expression vector 2 groups have higher coagulation activity, that is, the first nucleotide sequence described in Example 2 is constructed in the upstream expression region of the first CMV promoter downstream of the vector, and the second nucleotide sequence described in Example 2 When constructed in the downstream expression region of the second CMV promoter downstream of the vector, the coagulation activity of the cell fermentation liquid supernatant is higher, that is, the yield is higher.

Mismatch rate detection

Under the same other conditions, the supernatant of the 293F cell transiently transfected and expressed fermentation broth was purified by ProteinA and detected by SDS-PAGE. The results are shown in Figure 4 and Table 3: The chimeric protein harvested from the expression vector group 2 has the highest purity This shows that the expression vector containing the dual CMV promoter is better than the expression vector containing the CMV+EF1α promoter combination.

Table 3 Test results of mismatch rate

Example 5 Chimeric protein 2 half-life experiment

Using the literature "Wang Qihan, Huaicong, Sun Ruilin, et al. Use the CRISPR system to efficiently and quickly construct a mouse model of hemophilia B[C]//The 9th National Congress of the Chinese Society of Genetics and Academic Symposium Abstracts Collection (2009 -2013)." To establish a mouse model with FIX-deficient coagulation dysfunction, and perform the following experiments.

Half-life test

(1) 20 mice with FIX-deficient coagulation dysfunction were used. Among them, 10 mice were injected with the title compound chimeric protein of Example 2 through the tail vein. 2. Ten mice were injected with ALPROLIX (purchased from Biogen Idec) through the tail vein. Company), the injection dose level: 5mg/kg.

(2) After injection, blood was taken from the tail vein of the mouse at 0.25h, 1h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h and 192h to detect the concentration of FIX factor in plasma. The experimental results are shown in Table 4 and Figure 5.

Table 4: FIX factor concentration in mouse plasma at different time points after the tail vein injection of chimeric protein 2 and ALPROLIX into FIX-deficient coagulation dysfunction mice

Time (hour)	Chimeric protein 2 (%)	ALPROLIX (%)
0.25	100.0	100.0
1	79.0	87.8
8	55.1	53.1
twenty four	46.6	27.3
48	16.2	10.3
72	10.4	6.4
96	10.4	4.1

120	7.4	2.8
144	7.3	2.0
168	4.4	1.4
192	4.6	1.2

The results showed that after injection of chimeric protein 2 and ALPROLIX into the tail vein of mice with FIX-deficient coagulation dysfunction, the concentration of FIX factor in the plasma of mice injected with chimeric protein 2 decreased more slowly, and the half-life of chimeric protein 2 was higher than that The half-life of the third generation coagulation factor Alprolix.

The present invention has been described by the above-mentioned embodiments, but it should be understood that the above-mentioned embodiments are only for the purpose of illustration and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above-mentioned embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications fall under the protection of the present invention. Within the range. The protection scope of the present invention is defined by the appended claims and their equivalent scope.

Claims (20)

1. A chimeric protein comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a coagulation factor, and a first Fc variant of the Fc domain of an immunoglobulin, the The second polypeptide chain comprises a second Fc variant of the Fc domain of the immunoglobulin, the first Fc variant and the second Fc variant comprise an FcRn binding site, wherein the first Fc The variant contains the following amino acid mutations: calculated from the first starting amino acid at the N-terminus of the Fc domain of the immunoglobulin, the amino acid at position 146 is serine, the amino acid at position 148 is alanine, and The amino acid at position 187 is valine, and the second Fc variant contains the following amino acid mutations: Calculated from the first starting amino acid at the N-terminus of the Fc domain of the immunoglobulin, the amino acid at position 146 is Tryptophan.
2. The chimeric protein of claim 1, wherein the immunoglobulin is IgG, preferably, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4; more preferably, the immunoglobulin is human IgG1, IgG2 , IgG3 or IgG4; further preferably, the immunoglobulin is human IgG1 or IgG2, even more preferably, the immunoglobulin of the first polypeptide chain and the second polypeptide chain are the same, more preferably the first polypeptide chain The immunoglobulin of the peptide chain and the second polypeptide chain is human IgG1.
3. The chimeric protein of claim 1, wherein the sequence of the first Fc variant is shown in SEQ ID NO. 1 and the sequence of the second Fc variant is shown in SEQ ID NO. 2.
4. The chimeric protein of claim 1, wherein the coagulation factor is coagulation factor IX, coagulation factor VIII, or coagulation factor VII, preferably coagulation factor IX, more preferably, coagulation factor IX is from human and non-human species Preferably, the non-human species is selected from mice, dogs, cats, rabbits, birds, cattle, sheep, pigs, horses, fish, frogs and other primates, preferably the blood coagulation factor is human coagulation factor IX, more preferably The sequence of coagulation factor IX is shown in SEQ ID NO.25.
5. The chimeric protein of claim 1, wherein the sequence of the first polypeptide chain is shown in SEQ ID NO. 3, and the sequence of the second polypeptide chain is shown in SEQ ID NO. 4.
6. The chimeric protein of claim 1, wherein the first Fc variant and the second Fc variant further comprise the following amino acid mutations: the first amino acid mutation from the N-terminus of the Fc domain of the immunoglobulin The calculation starts with the starting amino acid, the amino acid at position 32 is tyrosine, the amino acid at position 34 is threonine, and the amino acid at position 36 is glutamic acid.
7. The chimeric protein of claim 1 or 6, wherein the sequence of the first Fc variant is shown in SEQ ID NO. 5, and the sequence of the second Fc variant is shown in SEQ ID NO. 6. Show.
8. The chimeric protein of claim 1 or 7, wherein the sequence of the first polypeptide chain is shown in SEQ ID NO. 7, and the sequence of the second polypeptide chain is shown in SEQ ID NO. 8. Show.
9. A pharmaceutical composition comprising the chimeric protein of any one of claims 1-8 and pharmaceutically acceptable excipients.
10. The chimeric protein of any one of claims 1-8, for use in the treatment of patients suffering from diseases that benefit from the administration of coagulation factors, or in the preparation of medicines for the treatment of patients suffering from diseases that benefit from the administration of coagulation factors the use of.
11. The use according to claim 10, wherein the disease is selected from coagulation disorders, bleeding disorders, hemophilia, or bleeding disorders.
12. The use according to claim 11, wherein the hemophilia is hemophilia B or hemophilia A, preferably, the hemophilia is hemophilia B.
13. A first nucleic acid molecule, said first nucleic acid molecule comprising a first nucleotide sequence encoding a first polypeptide chain of the chimeric protein of any one of claims 1-8;

    Preferably, the first nucleotide sequence is shown in SEQ ID NO. 9;

    Further preferably, the first nucleotide sequence is shown in SEQ ID NO.11.
14. A second nucleic acid molecule comprising a second nucleotide sequence encoding a second polypeptide chain of the chimeric protein of any one of claims 1-8;

    Preferably, the second nucleotide sequence is shown in SEQ ID NO. 10;

    Further preferably, the second nucleotide sequence is shown in SEQ ID NO.12.
15. An expression vector for expressing the chimeric protein according to any one of claims 1-8, wherein the expression vector comprises the first nucleic acid molecule according to claim 13 and the second nucleic acid molecule according to claim 14.
16. The expression vector according to claim 15, which comprises two promoters; preferably the expression vector comprises two identical promoters; preferably the two promoters are CMV promoter, EF1α promoter, or both Preferably, the expression vector contains two CMV promoters.
17. The expression vector of claim 15, wherein the first nucleotide sequence is shown in SEQ ID NO. 9, and the second nucleotide sequence is shown in SEQ ID NO. 10;

    Preferably, the first nucleotide sequence is shown in SEQ ID NO. 11, and the second nucleotide sequence is shown in SEQ ID NO. 12.
18. 17. The expression vector of any one of claims 15-17, wherein the first nucleotide sequence is upstream of the second nucleotide sequence.
19. A host cell comprising the expression vector of any one of claims 15-18.
20. The host cell according to claim 19, which is a mammalian cell, preferably, the host cell is a CHO cell.

Images