JP4451514B2 - Blood coagulation factor VII variant - Google Patents

Description

【００２９】
《実施例６．活性化された各改変体の調製》
精製した各改変体を、50mM Tris, pH 7.45, 0.1M NaClに透析し、ＦＸａを1/100（モル比）加え、50mM Tris, pH 7.45, 0.1M NaCl, 0.1％ PEG 8000, 100μg/mLリン脂質（Platerin(登録商標) Organotecnica社）、 10mM Ca²⁺、 37℃の条件下、１〜60分でインキュベーションし活性化した。活性化後、50mM Benzamidine-HClを加えて反応を停止し、抗ヒトＦＶＩＩモノクローナル抗体カラムで精製した（実施例４と同じ方法）。精製済みの各活性化改変体はTBS pH 8.0（0.1% PEG 8000含有）に透析し、-80℃に凍結保存した。活性化の程度は、ＳＤＳ−ＰＡＧＥで確認した。
【００３０】
《実施例７．活性化された各改変体の合成基質に対する水解活性測定》
実施例６に従い活性化された改変体ＶＩＩａ−３１を0.1μＭになるまで50mM Tris-HCl, 100mM NaCl,10mM Ca²⁺, 0.1% PEG 8000, pH 8.0で希釈し、そこに種々の合成基質を最終濃度1.0mMになるように加え、最終容量を200μｌとし、30℃で反応させ、１分間当たりの基質の水解量を見た。温度制御が可能な microplate reader Spectra max plus (Molecular device社）でpNAの遊離を405nmによる発色度として測定した。この結果を表２に示す。本発明の改変体の一つであるＶＩＩａ−３１は、何れの合成基質を対しても野生型（ＶＩＩａ−Ｗ）より高い水解活性を示し、その範囲は２〜２３倍であった。
【００３１】
【表２】

【００３２】
【発明の効果】
このように本願発明により得られたＦＶＩＩ及び／またはＦＶＩＩａの改変体は、野生型のＦＶＩＩに比べて明らかに高い酵素活性を有するものである。従って、本願発明の改変体は、血友病インヒビター患者への補充療法として極めて有効な薬剤となりうるものである。
【配列表】

【図面の簡単な説明】
【図１】 ＦＶＩＩの一次構造及び改変部位（星印）を示す図。
【図２】 ＦＶＩＩのプロテアーゼドメインアミノ酸配列を基にしたセリンプロテアーゼの基本構造を示す図。
【図３】 Ｘ線立体構造既知の各種トリプシン族セリンプロテアーゼ間の３Ｄマルチアライメントを示す図。
【図４】 野生型ＦＶＩＩ（ＦＶＩＩ−Ｗｉｌｄ）及び各種ＦＶＩＩ改変体のアミノ酸配列の一部を示す図。本図はＦＶＩＩの１５３番目のイソロイシンよりＣ末側のアミノ酸配列のみ示したもので、１５２番目のアルギニンよりＮ末側のアミノ酸配列についてはいずれも改変は行っておらず野生型と同じである。
【図５】 ＦＶＩＩ改変体作製用プライマー配列を示す図。
【図６】 ＦＶＩＩ改変体発現ベクターの構築方法を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a variant of blood coagulation factor VII (hereinafter sometimes referred to as FVII) and / or activated blood coagulation factor VII (hereinafter sometimes referred to as FVIIa) having enhanced enzyme activity. It is. Specifically, the present invention relates to a modified FVII / FVIIa whose activity is enhanced by substitution / deletion of an amino acid sequence unique to FVII, a pharmaceutical composition containing the modified as an active ingredient, and the pharmaceutical The present invention relates to a drug effective for treating a hemophilia inhibitor patient comprising the composition.
[0002]
[Prior art and problems to be solved]
FVII is a vitamin K-dependent blood coagulation factor and is widely known to be an initiation factor for exogenous blood coagulation. Similar to other vitamin K-dependent coagulation factors, the amino acid sequence from the N-terminal to 35 residues has a Gla region consisting of 10 gamma carboxyglutamic acids (hereinafter sometimes referred to as Gla) (Proc. Natl. Acad. Sci. USA, vol. 83, p. 2412-2416, 1986). FVII is in vitro activated blood coagulation factor X (hereinafter sometimes referred to as FXa), activated blood coagulation factor IX (hereinafter sometimes referred to as FIXa) or thrombin (hereinafter referred to as FIXa). It is known that 152Arg-153Ile is hydrolyzed and converted into an active FVII or FVIIa composed of an H chain and an L chain crosslinked by one S—S bond (J Biol. Chem., Vol. 251, p. 4797-4802, 1976).
[0003]
The enzyme activity of FVIIa itself is extremely weak and increases dramatically when it binds to the coenzyme tissue factor (TF) (Komiyama et al., Biochemistry, 29 (40), pp.9418-25 (1990)). The primary structure of FVIIa and TF, the crystal structure of the complex, and the binding site between the two molecules are also known at the amino acid residue level, but details of the catalytic activity amplification mechanism (conformational changes associated with TF binding) Is still unknown (Banner et al.,, Et al., Nature 380 (6569): pp. 41-6 (1996)).
[0004]
As replacement therapy for patients with hemophilia A and hemophilia B, administration of a blood coagulation factor VIII (hereinafter sometimes referred to as FVIII) and blood coagulation factor IX (hereinafter sometimes referred to as FIX) preparation is performed. It is done. However, the emergence of neutralizing antibodies (sometimes referred to as inhibitors) against FVIII and FIX has been regarded as a problem with the treatment.
[0005]
As a coping therapy for hemophilia patients who have produced such inhibitors, (1) FVIII factor overdose, (2) porcine FVIII factor administration, (3) complex preparations consisting of FII, FVII, FIX and FX Administration, (4) administration of FVIIa preparation, and the like. However, these methods are different in (1) for worsening symptoms by induction of higher titer inhibitors, (2) for antigenic shock, (3) for thrombus / DIC induction, (4 ) Has problems such as insufficient therapeutic effect and high cost due to large doses and frequent administration. Among these, when considering the balance between the effect and the risk, the most efficient one is administration of the FVIIa preparation of (4). However, since the FVIIa preparation is weak in activity, in order to exert the hemostatic effect, as described above, a large dose and frequent administration are required, and the treatment cost is greatly increased. In addition, the therapeutic effect is not sufficient as compared with the conventional replacement therapy performed for hemophilia patients.
[0006]
As a means for solving this problem, it is possible to prepare a modified form of FVII with increased enzyme activity, which is generally known to be difficult (Introduction to protein structure, Katsukabe). Supervised by Kouki et al., Published by an educational company, 1992). In particular, it is considered difficult to enhance the activity of blood coagulation factors by modification for the following reasons.
[0007]
Hemophilia is an abnormality of blood coagulation factors, and it is classified into two types: decreased activity due to quantitative deficiency and decreased activity due to qualitative abnormality. Many of these qualitative abnormalities are known to be (point) mutations, and as a result of analysis of hemophilia B patients with FIX abnormalities, molecular abnormalities exist throughout the FIX structure. It is clear that there are many examples in which the activity is 1% or less by replacing only one amino acid. Therefore, it is clear that even if the blood coagulation factor is modified unnecessarily, the activity is reduced.
[0008]
Moreover, according to the information obtained by Alanine Scannning (Dickinson et al., Proc. Natl. Acad. Sci. USA, 93 (25), pp. 14379-84 (1996)), 112 Alanine substitutions of FVII There is only one body that has increased enzyme activity, and no regularity has been found.
[0009]
As another attempt, Hopfner et al. Produced a modified FIX fragment with increased synthetic substrate activity using a method in which structural units composed of several amino acid residues of some domains constituting FIX are deleted or replaced. (EMBO J, 16 (22), pp. 6626-35 (1997)). However, this is not an intact FIX, but a partial fragment of FIX expressed in Escherichia coli and only has a synthetic substrate activity, so that it does not have blood coagulation activity as well as blood coagulation activity. It is. Furthermore, this is related to FIX, and does not suggest anything for FVII, which is another substance with completely different structure and specificity, and no report has been made so far regarding a variant with enhanced FVII enzyme activity. Absent.
[0010]
As described above, it has been considered that production of a modified body having a strong enzyme activity is difficult particularly for blood coagulation factors. Although attempts have been made to increase the synthetic substrate activity of the partial fragment in FIX, there has been no report on a variant of a blood coagulation factor having a high enzyme activity as an intact molecule.
[0011]
Therefore, the problem to be solved by the present invention is to produce and provide FVII and / or FVIIa having strong activity effective for the treatment of patients with hemophilia inhibitors in a situation where it is generally considered difficult to modify blood coagulation factors. It is to be.
[0012]
[Means for Solving the Problems]
Under the circumstances as described above, the present inventors have intensively studied to produce FVII having high enzyme activity, and have conducted various studies. As a result, the present invention has been completed. The present invention compares the amino acid sequence structure of FVII with various serine proteases, clarifies the amino acid sequence site unique to FVII, and deletes and substitutes the unique site for FVII with enhanced activity and / or It succeeded in producing the FVIIa variant.
[0013]
[Structure of the invention]
The basic structure of a group of serine proteases similar to the trypsin family consists of about 250 residues, and is roughly divided into two domains, the first half and the second half, on the amino acid sequence (FIG. 1). There are 6 β-strands in each domain, and they are formed as a structure having a total of 12 β-strands as proteases (FIG. 2). These 12 β-strands are so-called serine protease backbone structures, and the loops or helix regions connecting the strands are responsible for protease activity such as substrate specificity and reactivity with cofactors. It is considered. Examples of serine proteases include various blood coagulation factors such as FII, FVII, FVIII, FIX and FX, thrombolytic enzymes such as plasmin, and digestive enzymes such as trypsin, chymotrypsin and elastase.
Therefore, amino acid sequence structures of various serine proteases including FVII were compared to identify a region characteristic of FVII (FIG. 3). Then, using these sites as targets for modification, and referring to the structure of other serine proteases, FVII amino acid sequences having high enzyme activity were prepared by deleting and replacing the amino acid sequence of FVII. These modifications will be described in detail.
[0014]
(A) 159 Cys-164 Cys bond-modified variant (a- (1)) 159 Cys and 164 Cys are substituted with amino acid residues other than Cys, whereby the 159 Cys-164 Cys-modified variant (VII-5) ).
As a specific example of this variant, those obtained by substituting Cys for alanine (Ala) are shown in SEQ ID NO: 3 or 4 in the sequence listing. Here, Ala was selected as an example of amino acid residues other than Cys used for substitution, but unless substitution gives a major obstacle such as inactivation of enzyme activity other than cleaving the Cys-Cys bond, Any amino acid can be selected.
[0015]
(A- <2>) By replacing 164Cys with an amino acid residue other than Cys and substituting 299th valine (299Val) with Cys, 159Cys-164Cys is cleaved, and a disulfide is formed between 159Cys and 299Cys. A variant (VII-6) in which a bond (159Cys-299Cys) was formed.
As a specific example of this variant, those substituted with Ala as an amino acid residue other than Cys are shown in SEQ ID NO: 5 or 6. Here, as for amino acid residues other than Cys used for substitution, other than Ala other than Ala unless the enzyme activity is deactivated other than by cleaving the 159Cys-164Cys bond by substitution as described above Amino acids can be selected.
[0016]
(B) an amino acid sequence constituting a loop structure (hereinafter also referred to as 99-loop) consisting of an amino acid sequence from the 233rd threonine (233Thr) to the 240th asparagine (240Asn) in FVII or a part thereof Are variants that have been replaced, added or deleted.
As shown in FIG. 3, this region includes an amino acid sequence interposed between β strand 5 and β strand 6 that are commonly present in serine proteases. It is preferable to substitute this region with an amino acid sequence corresponding to the structure of another trypsin serine protease. A suitable example of a trypsin serine protease is human trypsin. Furthermore, as a specific example, the amino acid sequence from the 235th valine (235Val) to the 239th threonine (239Thr) in the 99-loop of FVII is an Asp-Arg-Lys-Thr in the trypsin loop structure. -Variant (VII-30) substituted with -Leu. This variant is described in SEQ ID NO: 7 or 8 in the sequence listing.
[0017]
(C) A variant in which the amino acid sequence constituting the intervening amino acid sequence from the 304th arginine (304Arg) to the 329th cysteine (329Cys) or a part thereof in FVII is substituted, added or deleted.
In particular, as shown in FIG. 3, in this region, FVII is several amino acid residues longer than other serine proteases in the amino acid sequence interposed between β-strand 8 and β-strand 9 that are commonly present in serine proteases. It is speculated that it can be a suitable target in FVII modification.
This region is preferably substituted with an amino acid sequence corresponding to the structure of another trypsin serine protease. A suitable example of a trypsin serine protease is human trypsin. Further, a preferred region that can be substituted, added or deleted in FVII constitutes a loop structure (sometimes referred to as 170-loop) consisting of the amino acid sequence from the 310th cysteine (310Cys) to the 329th cysteine (329Cys). Amino acid sequence or part thereof. Furthermore, as a specific example, the amino acid sequence from the 311st leucine (311Leu) to the 322nd asparagine (322Asn) in 170-loop of FVII is Glu-Ala-Ser- The modified body (VII-31) substituted by Tyr-Pro-Gly-Lys is mentioned. This variant is described in SEQ ID NO: 9 or 10 in the sequence listing.
[0018]
Furthermore, the above modifications (a) to (c) can be appropriately combined. As a specific example, for example, the combination of (b) and (c), that is, the amino acid sequence from the 235th valine (235Val) to the 239th threonine (239Thr) in the 99-loop of FVII is human trypsin. The amino acid sequence from Asp-Arg-Lys-Thr-Leu in the loop structure and the amino acid sequence from the 311st leucine (311Leu) to the 322nd asparagine (322Asn) in 170-loop is the loop structure of trypsin. The modified body (VII-39) substituted by Glu-Ala-Ser-Tyr-Pro-Gly-Lys inside is mentioned. This variant is described in SEQ ID NO: 11 or 12 in the sequence listing.
[0019]
The above-mentioned modified form can be obtained using a gene recombination method. The expression host is preferably a eukaryotic cell such as an animal cell. The variant of the present invention is obtained by incorporating cDNA encoding the amino acid sequence of each variant into an appropriate expression vector, transfecting the host cell, cloning the cell expressing the target gene, and obtaining the stable It is obtained by culturing and then purifying the expression strain.
[0020]
The FVII variant of the present invention can be used as an activated FVII (FVIIa) variant after various chemical treatments and the like.
[0021]
The FVII / FVIIa variants of the present invention can be formulated into pharmaceutical formulations for therapeutic, diagnostic or other uses. For formulations for intravenous administration, the composition will usually include a physiologically compatible substance, such as sodium chloride, glycine, etc., and have a buffered pH that is compatible with physiological conditions. Dissolve in aqueous solution. From the viewpoint of ensuring long-term stability, it may be considered to take the form of a lyophilized preparation as the final dosage form. It should be noted that guidelines for intravenously administered compositions are established by government regulations, such as “Biological Formulation Standards”.
Specific uses of the pharmaceutical composition comprising the FVII / FVIIa variant of the present invention include treatment of hemophilia inhibitor patients who have produced inhibitors of the blood coagulation factor by FVIII or FIX replacement therapy.
[0022]
【Example】
The present invention is illustrated by examples, but these examples do not limit the present invention. The present invention is illustrated in specific embodiments with reference to the accompanying drawings. In the examples, the modified product was expressed in the culture supernatant of animal cells (CHO-K1). Unless otherwise specified, reagents used for gene recombination were those manufactured by Takara Shuzo, Toyobo, and Perkin Elmer Applied New England Biolabs.
[0023]
Example 1 Cloning of FVII cDNA >>
A human liver cDNA library (Takara Shuzo) was purchased, and a FVII synthetic DNA sense primer to which a SalI site was added based on a known cDNA sequence (described in SEQ ID NO: 1 in the sequence listing) in literature (Molecular Basis of Thrombosia and Hemostasis) ( PCR was performed using VII-PWN; GGGGTCGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTG) and an antisense primer added with a BamHI site (VII-PWC; CCCGGATCCCTAGGGAAATGGGGCTCGCAGGAGGACTCCTGGGCG) and cloned into a commercially available cloning vector pCRII (Invitrogen). At this time, DNA sequencing was performed by a conventional method, and it was confirmed that it had a sequence known in the literature (Hagen FS et al, PNAS 1986; 83; 2412-6).
[0024]
Example 2 Preparation of FVII expression vector >>
The expression vector pCAGn (Japanese Patent No. 2824434) was digested with SalI and BamHI, and the DNA fragment prepared in Example 1 above containing the sequence encoding FVII was cut with SalI and BamHI, and ligated. And cultured on an LB agar medium containing ampicillin to select transformed E. coli. The emerged colonies were cultured overnight in a commercially available medium, and the target expression plasmid was extracted and purified to prepare “pVII-W”. The expression vector was sequenced and confirmed to have the target gene sequence.
[0025]
<< Example 3. Preparation of modified expression vector >>
Each FVII variant having the amino acid sequence shown in FIG. 4 was prepared by the following method. FIG. 4 shows only the amino acid sequence on the C-terminal side from the 153rd isoleucine of FVII. None of the amino acids on the N-terminal side from the 152nd arginine is the same as the wild type without modification. is there. PCR is performed using the synthetic DNA primer shown in FIG. 5 and the FVII gene as a template to obtain each amplified fragment. Each amplified fragment and the expression vector pCAGn cut with SalI and BamHI were ligated, transformed into E. coli JM105, cultured on LB agar medium containing ampicillin, and transformed E. coli was selected. The emerged colonies were cultured overnight in a commercially available medium, and the target expression plasmid was extracted and purified to prepare “pVII-5”, “pVII-30”, and “pVII-31” (FIG. 6). For “pVII-6”, PCR should be performed using the gene obtained using the primers (5) and (6) shown in FIG. 5 as a template and further using the primers (7) and (8). Was obtained. In addition, “pVII-39” was obtained by performing PCR using the gene obtained using the primers (9) and (10) as a template and further using the primers (11) and (12). Further DNA sequencing was performed to confirm that these plasmids had the target sequence.
[0026]
Example 4 Expression and purification of each variant in the culture supernatant >>
The above expression vector was used to transduce CHO cells with a commercially available lipofectin reagent, selected with G418 (1 mg / ml), and cells expressing the target gene were cloned by the limiting dilution method. Confirmation of the expression of the FVII variant was performed using a commercially available ELISA kit for FVII (Acerachrome FVII; Diagnostica Strago). The obtained stable expression strain was cultured in a serum-free medium (ASF104, Ajinomoto, penicillin, streptomycin, 20 μg / ml vitamin K, 1 mM butyric acid) and purified with an anti-human FVII monoclonal antibody column (Japanese Patent No. 2824430). Equilibration / washing and elution are performed using equilibration / washing buffer (50 mM Tris, pH 7.2, 0.1 M NaCl, 50 mM Benzamidine-HCl, 2 mM Ca ²⁺ ), elution buffer (50 mM Tris, pH 7.2, 0.1 M NaCl, 50 mM Benzamidine). -HCl, 10 mM EDTA). The purified variant was subjected to Western blotting using SDS-PAGE or a commercially available antibody against FVII to confirm that it was a FVII variant.
[0027]
<< Example 5. Measurement of coagulation activity of each variant >>
The clotting activity of each variant was measured by a clotting method using FVII-deficient plasma according to a conventional method. Each purified variant is diluted with Tris-BSA to 50 to 5 ng / ml, mixed with an equal volume with FVII deficiency, heated at 37 ° C. for 3 minutes, and relipidated TF (thromboplastin; Dade) Was added in an equal amount to initiate the coagulation reaction. The clotting time was measured, and the clotting activity was determined from the standard curve and dilution rate. The results obtained by converting the coagulation activity per protein concentration (measured by the Bradford method) and determining the specific activity are shown in Table 1. As a result, it was revealed that the FVII variant of the present invention has a clotting activity 2 to 6 times higher than that of plasma-derived FVII and wild-type recombinant FVII.
[0028]
[Table 1]

[0029]
Example 6 Preparation of each activated variant >>
Each purified variant is dialyzed against 50 mM Tris, pH 7.45, 0.1 M NaCl, FXa is added to 1/100 (molar ratio), and 50 mM Tris, pH 7.45, 0.1 M NaCl, 0.1% PEG 8000, 100 μg / mL phosphorous is added. Lipid (Platerin (registered trademark) Organotecnica), 10 mM Ca ²⁺ , incubated at 37 ° C. for 1 to 60 minutes for activation. After activation, 50 mM Benzamidine-HCl was added to stop the reaction, and purification was performed with an anti-human FVII monoclonal antibody column (the same method as in Example 4). Each purified modified variant was dialyzed against TBS pH 8.0 (containing 0.1% PEG 8000) and stored frozen at −80 ° C. The degree of activation was confirmed by SDS-PAGE.
[0030]
<< Example 7. Measurement of hydrolytic activity of each activated variant against synthetic substrate >>
Variant VIIa-31 activated according to Example 6 was diluted with 50 mM Tris-HCl, 100 mM NaCl, 10 mM Ca ²⁺ , 0.1% PEG 8000, pH 8.0 to 0.1 μM, where various synthetic substrates were added. The final concentration was 1.0 mM, the final volume was 200 μl, the reaction was carried out at 30 ° C., and the amount of substrate hydrolyzed per minute was observed. Using a microplate reader Spectra max plus (Molecular device) capable of controlling the temperature, the release of pNA was measured as the degree of color development at 405 nm. The results are shown in Table 2. VIIa-31, one of the variants of the present invention, showed higher hydrolytic activity than wild type (VIIa-W) against any synthetic substrate, and the range was 2 to 23 times.
[0031]
[Table 2]

[0032]
【The invention's effect】
Thus, the FVII and / or FVIIa variant obtained according to the present invention has a clearly higher enzyme activity than wild-type FVII. Therefore, the variant of the present invention can be an extremely effective drug as replacement therapy for hemophilia inhibitor patients.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the primary structure of FVII and modified sites (stars).
FIG. 2 is a diagram showing the basic structure of a serine protease based on the FVII protease domain amino acid sequence.
FIG. 3 shows 3D multi-alignment between various trypsin serine proteases with known X-ray conformations.
FIG. 4 is a diagram showing a part of amino acid sequences of wild-type FVII (FVII-Wild) and various FVII variants. This figure shows only the amino acid sequence at the C-terminal side from the 153rd isoleucine of FVII. The amino acid sequence at the N-terminal side from the 152th arginine is not modified and is the same as the wild type.
FIG. 5 is a view showing primer sequences for preparing FVII variants.
FIG. 6 is a view showing a method for constructing an FVII variant expression vector.

Claims (6)

The amino acid sequence from the 311st leucine (311Leu) to the 322nd asparagine (322Asn) in 170-loop of blood coagulation factor VII (hereinafter referred to as FVII) or activated blood coagulation factor VII (hereinafter referred to as FVIIa) FVII or a variant of FVIIa, characterized by being substituted with Glu-Ala-Ser-Tyr-Pro-Gly-Lys .   The variant according to claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 9 or 10 in the sequence listing. Additionally, the amino acid sequence from the 235th valine (235Val) to the 239th threonine (239Thr) in the 99-loop of FVII is substituted with Asp-Arg-Lys-Thr-Leu. Item 3. The variant according to Item 1 or 2 . The variant according to claim 3 , comprising the amino acid sequence of SEQ ID NO: 11 or 12 in the sequence listing. The pharmaceutical composition which contains the modified body in any one of Claim 1 to 4 as an active ingredient. A drug effective for the treatment of a hemophilia inhibitor patient comprising the pharmaceutical composition of claim 5 .

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