WO1991000291A1

WO1991000291A1 - Serpin variants

Info

Publication number: WO1991000291A1
Application number: PCT/EP1990/001026
Authority: WO
Inventors: Reinder Dijkema; Arie Visser
Original assignee: Akzo N.V.
Priority date: 1989-06-26
Filing date: 1990-06-23
Publication date: 1991-01-10
Also published as: AU5939390A; IE902276A1; ZA904930B; IE902276L

Abstract

Modified antithrombin III variants are developed which contain amino acid substitutions in the region comprising amino acids 384-396. In this region one or more amino acids are replaced by the corresponding amino acid(s) grouped around the factor Xa cleavage site in factor II related to the formation of meizothrombin. These amino acid substitutions result in altered heparin-dependent inhibitory effect of the modified antithrombin III vis-à-vis the factors IIa and Xa, respectively.

Description

Serpin variants

The invention is concerned with a modified antithrombin III (ATIII) polypeptide, a polynucleotide sequence coding therefore, a recombinant vector containing such a polynucleotide sequence, a method for the preparation of said ATIII polypeptide, as well as a pharmaceutical product containing the modified ATIII polypeptide.

Blood clotting is a dynamic process wherein a great number of enzymes and factors cooperate ultimately leading to a conversion of the fibrinogen circulating in the blood into a network of fibrin aggregates.

These blood clotting enzymes are activated consecutively and under each others action. The conversion of fibrinogen into fibrin takes place under direct action of thrombin (factor Ila) , which in turn is formed from prothrombin (factor II) , under the action of an enzyme complex containing among others factor Xa. Both factor Ila and factor Xa belong to the enzyme class serine endopeptidases. The activities of these factors leading to the conversion of fibrinogen into fibrin in turn are balanced by the so-called serine proteinase inhibitors (serpins) . One of the most well-known serpins is antithrombin III, a protein able to bind to both factor Ila and factor Xa and then form a stabile enzyme- pseudosubstrate complex with these respective factors. The formation of such a complex is stimulated by heparin, and leads to reduction of the amounts of factor Ila and factor Xa available for fibrinogen conversion.

This stimulation is the basis of the therapeutic use of heparin. However, a disadvantage of the use of heparin is that both factors Ila and Xa are inactivated to an about equal extent. This results in a relatively small therapeutic margin for heparin. Inhibition of one of the coagulation factors would improve this therapeutic margin. Therefore the present invention is concerned with modified ATIII polypeptides that in a heparin-dependent way can specifically inactivate either of the two enzymes.

A modified ATIII polypeptide according to the invention is characterized by an amino acid sequence which differs from the sequence of natural mature ATIII protein (represented in figure 1 as cDNA sequence) to such an extent that at least one of the amino acids in the region comprising amino acids no. 384-396 is replaced by the corresponding amino acid(s) grouped around the factor Xa cleavage-site in prothrombin (factor II) related to the formation of meizothrombin. These corresponding amino acids of factor II are represented in figure 2, together with the indicated polypeptide region of ATIII. Advantageously, one or more of the amino acids no. 384-389, 394, and 396 of ATIII are substituted according to the following scheme:

Ala³⁸⁴ -> Glu _Ser385 _ _Leu _τhr386 _→ L_Q..

Ala³⁸⁷ -> Glu

Val³⁸⁸ → Ser

Val³⁸⁹ → Tyr

Ala³⁹¹ → Asp

Ser³⁹⁴ -> He

Asn³⁹⁶ → Glu Modified ATIII polypeptides according to the invention are exemplified in figure 2 as mutants 1-10.

The abovementioned substitutions result in remarkable shifts in the biochemical characteristics of the polypeptide. In the presence of heparin the mature unmodified ATIII shows irreversible inhibition of the activities of both factors Ila and Xa. In contrast, polypeptides according to the invention wherein at least one of the amino acids no. 384-389 and 391 is replaced belong to the so-called type Ila inhibitors: in the presence of heparin they show a conserved irreversible inhibition of factor Ila, whereas their irreversible inhibition of factor Xa is lost (figure 3) .

On the other hand, polypeptides wherein at least one of the amino acids no.391, 394 and 396 is replaced belong to the so-called type Xa inhibitors: in the presence of heparin they show a conserved irreversible inhibition of factor Xa, whereas their irreversible inhibition of factor Ila is lost (figures 3 and 4) . In particular, a single amino acid substitution at position 394 can be solely responsible for the conservation of irreversible inhibition of factor Xa and the loss of irreversible inhibition of factor Ila. Modified ATIII polypeptides according to the invention are exemplified in figure 5 as mutants 11-18. The size and/or hydrofilicity of the amino acid at position 394 determines the heparin-dependent inhibition profile of the ATIII polypeptides (figure 6) .

Hence, it has been found that substitution in ATIII of the amino acids in the region 384-396 offers the possibility to specifically modulate the heparin- dependent activity of the polypeptide.

The ATIII variants according to the present invention can be applied therapeutically to influence blood clotting disorders, more specifically e.g. deep venous thrombosis, disseminated intravascular coagulation, and septic shock by either i.v. or s.c. route. The polypeptides according to the invention can be prepared with the aid of recombinant DNA technology. Herein a polynucleotide, coding for such a polypeptide is brought to expression. Such a polynucleotide can be prepared e.g. by modifying a polynucleotide sequence coding for ATIII by nucleotide substitutions in such a way that the resulting polynucleotide codes for a polypeptide according to the present invention. Another possibility is to synthesize a polynucleotide coding for the desired polypeptide, using known polynucleotide synthesis techniques. It goes without saying that both techniques mentioned also can be combined, resulting in a polynucleotide consisting partially of natural and partially of synthetic segments.

The polynucleotide either may code for the continuous stretch of the desired polypeptide or may code for two or more exons (each coding for a segment of the desired amino acid sequence) interrupted by one or more so-called introns (non-coding parts, which are excised during the process of expression of the polypeptide) . Of course, such a polynucleotide containing exons can only be used successfully in eukaryotic cells.

The abovementioned polynucleotides according to the invention are transferred to a suitable prokaryotic or eukaryotic, advantageously a mammalian, host cell by first incorporating the polynucleotide into a vector and then transforming the host with this recombinant vector. In this transformed host the polynucleotide is located under the control of other polynucleotide sequences which regulate expression and where appropriate secretion of the polypeptide product.

The polynucleotide can be present in the host in an autonomously replicating vector, or can be stably integrated in the genetic material of the host. It goes without saying that instead of the said modified AT III polypeptides use can be made also of smaller polypeptides containing the region of AT III essential for inhibition of factor Ila or factor Xa and containing the amino acid substitution(s) mentioned above. These polypeptides can be prepared by recombinant DNA technology or by conventional peptide synthesis.

Example METHODS

RNApreparation

Total RNA was extracted from either human fetal or adult liver samples by the guanidinium-phenol method (Chirgwin et al. Biochemistry lg, 5294, 1979) followed by oligo (dT) cellulose chromatography for selection of poly A⁺ mRNA (Maniatis et al. , Cold Spring Harbor Laboratory "Molecular Cloning: A Laboratory Manual", 1982).

cDNA synthesis and cloning

Complementary DNA was prepared according to a published procedure (Gubler and Hoffman, Gene 25, 263, 1983), provided with Eco RI linkers (Pharmacia), and ligated into phage λgtlO (Promega) . In vitro packaging, titration of recombinant phage in E. coli BNN102, and preparation of library DNA onto nitrocellulose filters was as described (Huynh et al. , DNA Cloning Techniques "A Practical Approach", 1984; Maniatis et al., ibid.). Clone identification

Oligodeoxynucleotides were synthesized using the phosphoramidite method on an Applied Biosystems 381A DNA synthesizer. A complementary probe was synthesized comprising a conserved region within human serine proteinase inhibitors (e.g. serpin antithrombin III: position 1358-1382 in figure 1) . Low stringency condi¬ tions for hybridization and washing were as described (Ragg, Nucl. Acids Res. 14, 1073, 1986). A partial cDNA clone was then used as nick-translated probe (Maniatis et al., ibid.) to obtain full-length cDNAs.

Site directed mut genesis

Oligonucleotide directed mutagenesis was performed using a cDNA fragment cloned in M13mpl8/19 as a template essentially as described (Nisbet and Beilharz, Gene Anal. Techn. 2, 23, 1985). The uracil containing phage M13mpl8/19 templates were prepared by their growth on E. coli RZ1032 (duf, ung^") as mentioned (Kunkel, Proc.Natl.Acad.Sci. USA £2, 488, 1985). Mutants were screened by determining their nucleic acid sequence (Sanger et al., Proc. Natl. Acad. Sci. USA 2t, 5463, 1977) .

Construction of expression plasmid

Wild type and variant antithrombin III genes were inserted in the unique restriction endonuclease BamHI site of the vector pKCR (0^!Hara et al., Proc. Natl. Acad. Sci. USA, 7§, 1527, 1981) in which the last exon region of the rabbit ,9-globin gene was removed by digestion with restriction endonuclease EcoRI and Bglll, filling in, and religation (position 1122-1196; Van Ooyen et al., Science 206. 337, 1979) and replacement of pBR322 for pBR327 sequences. Mammalian cell expression and affinity purification

For transient expression recombinant constructs were introduced by the DEAE-chloroquine method (Luthman and Magnusson, Nucl. Acids Res. 5_, 1295, 1983) in COS cells (Gluzman, Cell 23, 175, 1982).

Serum-free culture supernatants derived 24-72 h posttransfection were concentrated by Amicon YM10 filtration and dialyzed against 0.05 mol/1 Tris-HCl pH 8.0, 0.1 mol/1 NaCl.

For stable transformation recombinant constructs and pSV2neo (Colbere-Garapin et al., J.Mol.Biol. 150. 1, 1981) were introduced by the calcium phosphate precipitation method (Graham and van der Eb, Virology 52. 456, 1973) in CHO cells. To select CHO transfectants for the stable integration and expression of the neo gene, the antibiotic G418 (Gibco) was added 24 hours post¬ transfection at a concentration of l mg/ml. After 14 days, production of ATIII by pools of G418-resistant colonies was monitored by an ATIII-specific ELISA using goat anti-human ATIII IgG (Sera Lab) , rabbit anti-human ATIII IgG (Dakopatts) , and sheep anti-rabbit IgG HRP conjugate. 2.5 x 10⁶ Cells were seeded and grown for 48 hours in medium containing 10% foetal calf serum. After washing serum-free culture supernatants were harvested after an additional 48 hours and concentrated/dialyzed as mentioned above. Low-affinity monoclonal antibody AATR-1 (Interferon Sciences) coupled to tresyl-activated Sepharose (Pharmacia) was added to the processed supernatant and incubated for 4 hours at room temperature. Elution of bound ATIII was performed for 10 minutes in a volume of 1 ml of 3 mol/1 NaSCN, 0.01 mol/1 Tris-HCl pH 8.0. Immediately after elution, samples were dialyzed against 0.05 mol/1 Tris-HCl pH 8.0, 0.1 mol/1 NaCl. The concentration of ATIII was determined by the above-mentioned ELISA. Inhibition assays

The heparin-dependent factor X_a and factor II_a inactivating potency of antithrombin III variants has been investigated with the help of the continuously monitored enzyme inactivation assay (Tian and Tsou, Biochemistry 21, 1028, 1982). They are expressed as the percentage of inhibition relative to the wild type ATIII.

RESULTS

- A recombinant phage containing part of the antithrombin III structural gene was obtained from a human fetal liver cDNA library using the conserved serpin probe. Its ATIII origin was confirmed by positive hybridization with a reactive site oligonucleotide probe (position 1253-1294 in figure 1) as well as DNA sequence analysis. The partial cDNA ATIIIcDNAl extends from posi¬ tion 887 to 1472. Two nucleotide differences were found when ATIIIcDNAl was compared to the ATIII cDNA reported by Bock et al. (Nucl. Acids Res. 10, 8113, 1982) ; C at position 1049 and T at position 1317 both differences are silent mutations not changing the corresponding amino acid codons. The insert of ATIIIcDNAl was used as a probe in subsequent screening of a human adult liver cDNA library resulting in ATIIIcDNA2 extending from position 75 to 1479 (Bock et al., ibid.). The incomplete ATIII leader sequence was replaced by a synthetic DNA leader derived from the published ATIII cDNA sequence (Bock et al., ibid.). In addition, upstream of the ATIII leader sequence a suitable restriction endonuclease EcoRI site was positioned at position -10 (5'-GAATTCCACCATG-3*) . The complete ATIII cDNA cassette therefore consists of the following regions: i EcoRI (position -10) towards SacII (position 130) derived from synthetic origin, ii SacII (position 130) towards Ncol (position 935) derived from ATIIICDNA2, and iii Ncol (position 935) towards EcoRI (position 1474) derived from ATIIIcDNAl. All fragments were ligated in the unique restriction endonuclease EcoRI site of pBR327.

- Inspection of the ATIII cDNA sequence around the reactive centre (P-region) indicate the existence of surrounding restriction endonuclease PvuII (position 1241) and Stul (position 1312) sites. Since an additional restriction endonuclease PvuII site is present at posi¬ tion 1096 this was deleted by site directed mutagenesis of residue G-QØI into

disturbing the restriction endonuclease PvuII recognition site, but still leaving the amino acid codon intact (CTG → CTT: Leu) . Mutations introduced in the reactive centre P-region of ATIII can be performed by the replacement of the internal PvuII - Stul fragment (position 1241 to 1312) by a synthetic P- region of choice.

- Mutations introduced into the ATIII reactive centre P-region are derived from the human prothrombin gene (Degen and Davie, Biochemistry 26, 6165, 1987). Prothrombin participates in the final stage of clot formation since it is activated to thrombin by factor X_a. Selection of prothrombin sequences known to be physiological substrates for factor X_a and integration of (parts of) these sequences within the ATIII reactive centre P-region influences the inhibition profile of ATIII between factor X_a and factor II_a. The different synthetic P-regions, being hybrids between ATIII and prothrombin sequences, are illustrated in figure 2; they were used to replace the wild type PvuII - Stul fragment of the original ATIII cDNA backbone in pBR327 and confirmed by DNA sequence analysis.

- ATIII variants are evaluated for their inhibition profile in the following way. The variant ATIII containing fragments are isolated by digestion with restriction endonuclease EcoRI and filling in by DNA polymerase. The blunt-ended fragments are then ligated in pKCR digested with restriction endonuclease BamHI and filling in by DNA polymerase. Correct orientation of the fragments with respect to the SV40 promoter was checked by restriction endonuclease mapping. The resulting recombinants were used for transient and stable expression in COS and CHO cells, respectively. Culture supernatants were tested for in vitro heparin-dependent inhibitory activity towards factor Xa and factor Ila. - The ATIII P-region variants possessing different amino acid substituents derived from the prothrombin sequence (i.e. factor Xa cleavage site related to the formation of meizothrombin) are summarized in figure 2. As shown in figures 3 and 4 substitutions introduced in this way result in a remarkable shift in the heparin-dependent inhibition profile of ATIII towards factors Ila and Xa. With respect to the ATIII variant class being specific for factor Xa this can be accomplished by two independent ways. Either modification of at least amino acid residue 394 (Ser³⁹⁴ → He) or modification of amino acid residues 391 and 396 (Ala³⁹¹ → Asp and Asn³⁹⁶ → Glu) result in a common inhibition profile of the modified ATIII. In addition, the nature of the amino acid substitution at position 394 is crucial in conferring inhibitory profile to the ATIII polypeptide (figures 5 and 6) . Therefor, other amino acid substitutions at position 394 that represent a large and/or hydrofilic character (Chotia, Ann.Rev.Biochem. 3_3, 537, 1984) predict a specificity of heparin-dependent inhibition towards factor Xa. Finally, modelling studies of factor Xa support the requirement of negatively charged residues within ATIII at the P3 and P3* position in order to form salt bridges with positively charged residues present in the S3 and S3¹ binding sites (Greer, J.Mol.Biol. 153. 1043, 1981). Figure 1;

Antithrombin III cDNA sequence of reconstructed cassette.

Asterisks indicate differences observed with the ATIII cDNA sequence of Bock et al. (ibid.). Broken underlined restriction endonuclease sites were used for reconstruction experiments.

Underlined restriction endonuclease sites were used for P region replacement studies. The nucleotide

indicted by 0 was mutagenized towards T-J QJ for deletion of the additional restriction endonuclease PvuII site.

The arrows indicate the start positions of partial ATIII cDNA clones obtained during this study.

Figure 2;

Antithrombin III P-region variants (PvuII-StuI: position

1244-1314)

Antithrombin III (ATIII; Bock et al., ibid)

Prothrombin (FII; Degen and Davie, ibid)

Amino acids that differ from the natural ATIII polypeptide are underlined.

Figure 3:

Heparin-dependent inhibition of factors Ila and Xa of ATIII and variants of culture supernatants derived from transfected COS cells. WT = ATIII, COS = COS control and T = Tris-buffer.

Figure 4/Figure 6:

Heparin-dependent inhibition of factors Ila and Xa of ATIII and variants after affinity purification of culture supernatants derived from transfected CHO cells. WT = ATIII, C = CHO control, CT = transfected CHO control and T = Tris buffer. Figure S ;

Antithrombin III P^ variants

Amino acids that differ from the natural ATIII polypeptide are underlined.

Claims

1. Modified antithrombin III polypeptide, characterized in that it differs from natural mature antithrombin III protein in that at least one amino acid from the region comprising amino acids 384-396 is replaced by the corresponding amino acid(s) grouped around the factor Xa cleavage site in factor II related to the formation of meizothrombin, or a fragment thereof.

2. Modified antithrombin III polypeptide or a fragment thereof according to claim 1, wherein at least one of the following amino acid substitutions has taken place:

Asn³⁹⁶ -> Glu

3. Modified antithrombin III polypeptide or a fragment thereof according to claim 1 or 2, wherein at least the following amino acid substitution has taken place: Ser³⁹⁴ → He.

4. Modified antithrombin III polypeptide or a fragment thereof according to claim 1 or 2, wherein at least the following amino acid substitution has taken place: Ala³⁹¹ → Asp.

5. Modified antithrombin III polypeptide or a fragment thereof according to claim 1 or 2, wherein at least the following amino acid substitution has taken place: Asn³⁹⁶ → Glu.

6. Modified antithrombin III polypeptide or a fragment thereof according to claim 4 or 5, wherein at least the following amino acid substitutions have taken place: Ala³⁹¹ → Asp and Asn³⁹⁶ → Glu.

7. Nucleic acid sequence at least part of which is coding for a modified antithrombin III polypeptide or a fragment thereof according to claim 1-6.

8. Nucleic acid sequence according to claim 7, furthermore comprising at the 5 terminus of the sequence coding for the polypeptide a signal sequence and/or a promoter.

9. Vector comprising a nucleic acid sequence according to claim 7-8.

10.Host cell containing a nucleic acid sequence according to claim 7-8 or a vector according to claim 9.

11.Host cell according to claim 10 characterized in that it is derived from a mammalian cell.

12.Pharmaceutical preparation containing a polypeptide or a fragment thereof according to claim 1-6 as well as a pharmaceutically acceptable carrier.