EP2162147A2

EP2162147A2 - Modified human factor vii/viia and pharmaceutical composition containing same

Info

Publication number: EP2162147A2
Application number: EP08805990A
Authority: EP
Inventors: Emmanuel Nony; Sami Chtourou; Nicolas Bihoreau
Original assignee: LFB Biotechnologies SAS
Current assignee: LFB Biotechnologies SAS
Priority date: 2007-06-15
Filing date: 2008-06-12
Publication date: 2010-03-17
Also published as: CN101743017A; FR2917414A1; JP2010529848A; WO2008155509A3; KR20100039281A; CA2691110A1; US20110162094A1; WO2008155509A9; AR067335A1; BRPI0813372A2; FR2917414B1; TW200906851A; WO2008155509A2; AU2008265021A1

Abstract

The invention relates to modified factors VII/VIIa having good stability, nucleic acids coding for such modified factors VII/VIIa and methods for producing same.

Description

TITLE OF THE INVENTION

Modified human factor VII / VIIa and pharmaceutical composition containing same.

FIELD OF THE INVENTION

The present invention relates to the field of the manufacture of factors VII

(FVI 1) activated human activated FVIIa (FVIIa) agents useful as active ingredients of drugs. In particular, the invention relates to modified Vll / VIIa factors having high stability, nucleic acids encoding such modified FVII / VIIa, and methods for their production.

PRIOR ART

FVII is a vitamin K-dependent glycoprotein that, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX in the presence of calcium and tissue factor. FVII is secreted as a unique peptide chain of 406 amino acid residues, which has a molecular weight of about 50 kDa. FVII comprises four distinct structural domains: the N-terminal γ-carboxylic (GI) domain, two epidermal growth factor like (EGF-like) domains, and a serine protease domain. Activation of FVII in FVIIa is characterized by cleavage of the Arg152-I153 linkage (Arginine 152-Isoleucine 153). The FVIIa is therefore composed of a light chain of 152 amino acids with a molecular weight of approximately 20 kDa and a heavy chain of 254 amino acids with a molecular mass of approximately 30 kDa bound together by a single disulfide bridge (Cysteine 135-Cysteine 262). FVII / Vlla is used in the treatment of hemophilia patients with factor VIII (haemophilia A) or factor IX (haemophilia B) deficiency, as well as patients with other factor deficiencies. coagulation, for example an inherited deficiency of FVII. FVII / Vlla is also recommended for the treatment of stroke. The oldest method of obtaining FVIIa concentrates was the purification of FVIIa from plasma proteins from fractionation.

For this purpose, the document EP 0 346 241 describes the preparation of a fraction enriched in FVIIa, obtained after absorption and elution of a by-product of the fractionation of plasma proteins containing FVII and FVIIa and other proteins such as proteins. factors IX, X and II, in particular the PPSB preeluatus (P = prothrombin or FII, P = proconvertin or FVII, S = Stuart factor or FX and B = antihemophilic factor B or FIX).

Similarly, EP 0 547 932 describes a method of manufacturing a high purity FVIIa concentrate essentially free of vitamin K dependent factors and FVIII. One of the major drawbacks of these methods of obtaining FVII / VIIa from blood plasma is that it allows only small amounts of product to be obtained. Moreover, a major disadvantage lies in the fragility of the products obtained which systematically have truncated forms and therefore less active and likely to cause undesirable side effects. In addition, the supply of plasma collected from blood donors remains limited.

Therefore, as early as the 1980s, DNA encoding human factor VII was isolated (Hagen et al (1986), Proc Natl Acad Sci USA, Apr 83 (8): 2412-6 ) and the corresponding protein expressed in BHK (Baby Hamster Kidney) mammalian cells (EP 0 200 421). The application FR 06 04872 filed by the Applicant also describes the production of FVIIa in a transgenic animal.

These production methods make it possible to obtain secure proteins in terms of contamination by viruses or other pathogens. Such methods make it possible to obtain proteins whose primary sequence is identical to the human primary sequence.

Commercial preparations of recombinant human FVIIa are currently available as NovoSeven ^® (NovoNordisk ™). Relatively high doses, as well as frequent intravenous administrations, are necessary to achieve and maintain the desired therapeutic or prophylactic effect. This treatment is therefore both constraining for the patients and very expensive.

It has also been shown that FVII / VIIa is a protein sensitive to proteolytic cleavage resulting in the formation of numerous degradation products devoid of coagulating activity (atypical cleavages). Atypical cleavages may occur at different stages of the production process but also during the storage of FVII / VIIa. Degradation products were observed for both plasma derived FVII / VIIa but also for genetically recombined FVII / VIIa. Atypical cleavages may occur prior to activation of FVII in FVIIa, for example during production and purification of FVII, during the activation step per se or during purification and / or storage of the activated product (FVIIa ). EP 0 370 036 relates to a modified FVII / VIIa on residues lysine, arginine, isoleucine and / or tyrosine involved in the atypical cleavage of FVII / VIIa in order to reduce the atypical cleavages of FVII / VIIa and thus obtain an FVII / VIIa more stable. However, this patent only partially solves the problem of obtaining a more stable FVII / VIIa since it does not address the problem of the alteration of the conformation of FVII / VIIa linked to the modification of the amino acids involved in atypical cleavage. This patent neither describes nor suggests the production of a modified FVII / VIIa at atypical cleavage sites whose conformation is not or only slightly altered by the modification of the amino acid sequence. Despite the existence of documents relating to modified human FVII / VIIa, particularly at atypical cleavage sites, there is still a great need for new human FVII / Vlla with improved properties.

SUMMARY OF THE INVENTION

The invention relates to FVII / Vlla factors having a high stability, modified on at least two amino acid residues selected from lysine 38, arginine 290 and arginine 315, said amino acid residues being ( i) substituted with a distinct amino acid residue or (ii) deleted. The subject of the invention is also nucleic acids encoding the factors

FVII / FVIIa modified above, recombinant vectors in which said nucleic acids are inserted, host cells transformed with said nucleic acids or said recombinant vectors and genetically modified organisms expressing said modified FVII / VIIa factors. The invention also relates to a method for producing a factor

FVII / FVIIa modified defined above.

The invention also relates to the use of the FVII / VIIa factors modified above for the manufacture of medicaments, as well as to pharmaceutical compositions comprising said modified FVII / VIIa factors.

DESCRIPTION OF THE FIGURES

Figure 1: MALDI-TOF mass spectrum under native conditions showing the amino acid sequences resulting from the atypical cleavage of FVII.

Figure 2: MALDI-TOF mass spectrum under reducing conditions showing the amino acid sequences resulting from the atypical cleavage of FVII.

Figure 3: Molecular modeling representing the superposition of native human FVII structures containing lysine 38 (blank) and modified human FVII containing glutamine at position 38 (in black) using Sybyl 7.2 software (Tripos).

Figure 4: Molecular modeling depicting the superposition of native human FVII structures containing arginine 290 (blank) and modified human FVII containing glutamine at position 290 (black) using Sybyl 7.2 software (Tripos).

Figure 5: Molecular modeling representing the superposition of native human FVII structures containing arginine 315 (in white) and modified human FVII containing glutamine at position 315 (in black) using Sybyl 7.2 software (Tripos).

DESCRIPTION OF THE INVENTION

Novel modified FVII / VIIa factors are provided by the present invention, which have high stability both (i) during storage time and (ii) in vivo after administration to patients. The Applicant has shown that, surprisingly, certain mutations of the amino acid residues lysine 38 (Lys 38, K38), arginine 290 (Arg290, R290) and arginine 315 (Arg315, R315) in the amino acid sequence of Natural human FVII / FVIIa do not alter or little the conformation of human FVII / Vlla thus modified, compared to a natural human FVII / Vlla.

In addition, the Applicant has shown that a modified FVII / VIIa according to the invention, whose three-dimensional conformation is very close to, and sometimes identical to, the three-dimensional conformation of natural human FVII / FVIIa, has improved properties, including a decreased atypical cleavage rate, improved production yields, decreased clearance, and increased stability compared to one of natural human FVII / VIIa, while maintaining a conformation similar to that of natural human FVII / VIIa.

By atypical cleavages is meant any cleavage of peptide bonds, excluding cleavage of the activation site (cleavage of the ATg ₁₅₂ -IIe _{I53 bond} ), involved in the FVII or FVIIa molecule. These atypical cuts relate in particular to the amino acids lysine 38 (Iysine38-leucine39 bond), arginine 290 (arginine290-glycine291 bond) and arginine 315 (arginine315-lysine316 bond) and cause structural modifications resulting in an alteration of the pharmacokinetic properties of the FVII / VIIa. By production yield is meant the amount of structurally consistent and active FVII / VIIa produced per volume of fermenter (or bio-reactor) or volume of milk from transgenic animals or mass of any biomass (animal cells, vegetable, bacterial or insect). The cost of producing a mutated FVII / VIIa is therefore significantly lower than a FVII / VIIa whose primary sequence is identical to the native human FVII / VIIa sequence.

By clearance, denotes the fraction of a theoretical volume totally purified, that is to say no longer containing FVII / VIIa per unit of time. The clearance of FVII / FVIIa represents a plasma clearance coefficient. This corresponds to the ability of an organ to completely eliminate FVII / FVIIa from a given volume of arterial plasma per unit of time. The clearance of FVII / FVIIa is the apparent (virtual) volume of arterial plasma completely cleared of FVII / FVIIa given per unit of time.

By stability is meant the ability of FVII / VIIa to maintain its chemical, physical, structural, conformational and / or biopharmaceutical properties throughout its validity. By conformation, is meant the tertiary structure of a protein, that is to say the folding of the polypeptide chain in space. We also speak of three-dimensional structure, or 3D structure. The conformation of a protein is intimately related to its biological activity, so when this structure is altered the protein loses its biological activity, it is denatured. By alteration of the conformation, we designate therefore any modification of the three-dimensional structure of a protein resulting in a loss of the biological activity of said protein.

The biological activity of FVII / VIIa of the invention can be quantified by measuring the ability of FVII / VIIa to induce blood coagulation by means of FVII deficient plasma and thromboplastin, as for example described in US Pat. 5997864. In the test described in US Pat. No. 5,997,864, the biological activity is expressed by a reduction in coagulation time relative to the control sample, and is converted to "units of FVII / VIIa" by comparison with a human serum standard. containing 1 unit / ml of FVII / VIIa activity. The FVII / VIIa of the invention has posttranslational modification characteristics similar to that of native human FVII / VIIa but may also have post-translational modifications different from those of native plasma human FVII / VIIa so as to enhance its properties. chemical, physical, structural, conformational and / or biopharmaceutical. In its broadest aspect, the invention relates to a human FVII / VIIa modified with respect to the peptide sequence of native human FVII / VIIa of which at least two amino acids selected from lysine 38, arginine 290 and arginine 315 are substituted or deleted, characterized in that

(i) lysine 38 is substituted with an amino acid selected from glutamine, alanine, glutamic acid, glycine, isoleucine, leucine, methionine, histidine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine, preferably glutamine, histidine or glutamic acid; (ii) arginine 290 is substituted with an amino acid selected from glutamine, alanine, glutamic acid, asparagine, glycine, isoleucine, leucine, methionine, histidine, phenylalanine proline, serine, threonine, tryptophan, tyrosine or valine, preferably glutamine, histidine, asparagine or glutamic acid, and / or

(iii) arginine 315 is substituted with an amino acid selected from glutamine, alanine, glutamic acid, asparagine, glycine, isoleucine, leucine, methionine, histidine, phenylalanine proline, serine, threonine, tryptophan, tyrosine or valine, preferably glutamine, histidine, asparagine or glutamic acid.

In a preferred embodiment of the present invention, FVII / VIIa contains at least two substitutions selected from glutamine substituted lysine 38, glutamine substituted arginine 290, and glutamine substituted arginine 315.

In a first particular embodiment of the present invention, FVII / VIIa contains a mutation on lysine 38 and arginine 290.

In a second particular embodiment of the present invention, FVII / VIIa contains a mutation on lysine 38 and arginine 315. In a third particular embodiment of the present invention, FVII / VIIa contains a mutation on arginine 290 and arginine 315.

In a fourth particular embodiment of the present invention, FVII / VIIa contains a mutation on lysine 38, arginine 290 and arginine 315. In a particular embodiment of the present invention, lysine 38 is substituted. by glutamine, arginine 290 is substituted by glutamine and arginine 315 is substituted by glutamine.

The FVII / VIIa of the present invention can be produced using recombinant DNA (or genetic recombination) technologies. In general, the nucleic acid sequence of a nucleic acid (DNA or RNA) encoding native human FVII / VIIa is modified to encode the desired protein, particularly a modified FVII / FVIIa according to the invention. The nucleic acid thus modified can then be inserted into an expression vector, which expression vector is then used to transform or transfect a host cell. A nucleic acid encoding a native or natural human FVII / VIIa is illustrated by the nucleic acid of sequence SEQ ID No. 1.

Thus, another object of the present invention relates to a nucleic acid encoding a modified human FVII / VIIa according to the present invention, as well as a nucleic acid of complementary sequence. A nucleic acid encoding a human FVII / VIIa modified according to the present invention. The invention may be produced or synthesized using any of the conventional techniques forming part of the general knowledge of those skilled in the art. By way of illustration, a nucleic acid encoding a modified human FVII / VIIa according to the invention may be made by genetic recombination from the nucleic acid encoding native human FVII / VIIa. Preferably, the nucleic acid encoding the modified human FVII / VIIa is obtained by site-directed mutagenesis from the nucleic acid encoding native human FVII / VIIa. Site-directed mutagenesis techniques are well known to those skilled in the art and provide a DNA encoding the desired modified human FVII / VIIa. In particular, site-directed mutagenesis techniques that are identical or derived from the site-directed mutagenesis technique described by Michael Smith in 1978 (Smith et al., "Mutagenesis at a Specified Position in a DNA Sequence"; J Biol Chem. (1978) Sep 25; 253 (18): 6551-60).

Advantageously, the FVII / VIIa of the invention is a polypeptide of which at least two amino acid residues selected from the amino acids lysine 38, arginine 290 and arginine 315 of native human FVII of sequence SEQ ID No. 2 are mutated by amino acids chosen by design, or are deleted.

In a particular embodiment, the modified FVII / VIIa of the invention can be obtained from a variant of native human FVII / VIIa, since this variant is not more immunogenic than human FVII / VIIa. native. Thus, the peptide sequence of this variant may have at least 70% identity, and advantageously at least 80% or 90%, and even more advantageously at least 99% identity. in amino acids with the peptide sequence of native human FVII and comprising at least two amino acid residues selected from the amino acids lysine 38, arginine 290 and arginine 315, according to the amino acid numbering of native human FVII SEQ ID sequence No. 2 are mutated by purposely selected amino acids, or are deleted. Such a variant has essentially the same or better biological activity as native human FVII / VIIa.

For purposes of this disclosure, the term "nucleotide sequence" may be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not restricted to information about its sequence.

The terms "nucleic acid", "polynucleotide", "oligonucleotide" or "nucleotide sequence" include RNA sequences, DNA sequences, cDNA sequences or hybrid RNA / DNA sequences of more than one nucleotide, regardless of in the single-stranded form or in the form of duplex. The term "nucleotide" refers to natural nucleotides [Adenine (A), Thymine (T), Guanine (G), Cytosine (C) and Uracil (U)].

For purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide whose orientation is reversed. The complementary "bases" are A with T (or A with U), and C with G.

According to the invention, a first nucleic acid having at least 90% identity with a second reference nucleic acid will have at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% nucleotide identity with said second reference nucleic acid. According to the invention, a first polypeptide having at least 90% identity with a second reference polypeptide, will have at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96% 97%, 97.5%, 98%, 98.3% 98.6%, 99%, 99.6% amino acid identity with said second reference polypeptide. The "percentage identity" between two nucleic acid sequences, or between two amino acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.

The part of the nucleotide sequence, or of the amino acid sequence, in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain an optimal alignment between the two sequences.

The percent identity is calculated by determining the number of positions at which an identical nucleotide base, or an identical amino acid, is observed for the two sequences compared, then dividing the number of positions at which there is identity between the two nucleic bases, or between the two amino acids, by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of identity in nucleotides, or in amino acids, of the two sequences between them.

The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms.

Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = "aligned"; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (1 1) PHYLOGENETIC TREE / TREE TYPE = "none"; (12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide".

The present invention also relates to an expression vector in which has been inserted a nucleic acid encoding a modified human FVII / VIIa according to the present invention.

The expression vector used in the present invention may contain a promoter capable of directing transcription of the nucleic acid encoding FVII / VIIa of the invention. Promoters widely used in mammalian cell cultures include viral promoters and cell promoters well known in the art. The expression vector may also contain splice sites located downstream of the promoter and upstream of the insertion site of the FVII / VIIa-encoding DNA sequence of the present invention. The expression vector may also contain a polyadenylation sequence located downstream of the insertion site of the DNA sequence encoding FVII / VIIa of the present invention. The expression vector may also contain any type of DNA sequence useful for the expression, selection and / or insertion of FVII / VIIa, the DNA sequence encoding FVII / VIIa of the present invention and / or expression vector containing the DNA sequence encoding FVII / VIIa of the present invention.

A further object of the present invention is a cell transformed to produce a modified human FVII / VIIa according to the present invention. These transformed cells are made by transferring a nucleic acid encoding a modified human FVII / VIIa according to the present invention into the genome of a host cell, preferably to express that DNA sequence by the thus transformed cell. Suitable techniques for cell transformation are well known to those skilled in the art. These techniques include, but are not limited to the use of liposomes, the use of polyethylene glycol (PEG), the use of DEAE-dextran, the use of calcium phosphate, the use of viruses (mainly retroviruses), the use of a cannon DNA, cell fusion, microinjection, electroporation etc. The invention therefore also relates to a cell transformed with a nucleic acid encoding a modified human FVII / VIIa defined above and expressing said modified human factor VII / VIIa. Preferably, said transformed cell consists of a mammalian transformed cell, and in particular a transformed rodent, a bovine, a goat, a porcine, a non-human primate or a non-human primate cell. human cell. The modified human FVII / VIIa according to the present invention can be obtained from a transformed cell according to the present invention and cultured. By way of example, mention may be made of the following cells: BHK (Baby Hamster Kidney) and in particular BHK tk ^" ts13 (CRL 10314, Waechter and Baserga, Proc Natl Acad Sci USA 79: 1 106-1 110, 1982), CHO (ATCC CCL 61), COS-1 (ATCC CRL 1650), HEK293 (ATCC CRL 1573, Graham et al., J. Gen. Virol 36: 59-72, 1977), Rat Hep I (Rat hepatoma ATCC CRL 1600), Rat Hep II (Rat hepatoma, ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (CHO cell line) ( Urlaub and Chasin, Proc Natl Acad Sci USA 77: 4216-420, 1980), YB2 / 0 cells, 3T3 cells, Namalwa cells, or BHK cells adapted for serum-free culture (US 6,903,069).

The present invention also relates to an organism genetically modified to produce a modified human factor VII / VIIa according to the present invention. According to the definition given by the European Union, a genetically modified organism is an organism (with the exception of human beings) whose genetic material has been modified in a way that can not be carried out naturally by multiplication and / or by recombination. With respect to the present invention, a genetically modified organism incorporates the FVII / VIIa-encoding DNA sequence of the present invention and expresses said modified human FVII DNA sequence to produce said modified human FVII / VIIa of the present invention. The genetically modified organism is a microorganism, an animal or a plant. The invention therefore also relates to a genetically modified organism comprising in its genome a nucleic acid encoding a modified human factor VII / VIIa as defined in the present description, and expressing said modified human factor VII / VIIa.

A microorganism is a microscopic organism, it can be a bacterium, a yeast or a virus. The bacterium may be, for example, Bacillus subtilis (Palva et al (1982) Proc Natl Acad Sci USA 79: 5582, EP 0 036 259 and EP 0 063 953, WO 84/04541); Escherichia coli (Shimatake et al (1981) Nature 292: 128, Amann et al (1985) Gene 40: 183, Studier et al (1986) J. Mol Biol 189: 13, EP 0 036 776, EP 0 136 829 and EP 0 136 907); Streptococcus cremoris (Powell et al (1988) Appl., Environ., Microbiol., 54: 655], Streptococcus lividans [Powell et al (1988) Appl. Microbiol. 54: 655); Streptomyces lividans (US 4,745,056). Yeast can be, for example, Candida (Kurtz et al (1986) Mol., CeII, Biol., 6: 142, Kunze et al (1985) J. Basic Microbiol., 25: 141); Hansenula (Gleeson et al (1986) J. Gen. Microbiol 132: 3459, Roggenkamp et al (1986) Mol Gen. Genet 202: 302); Kluyveromyces (Das et al., (1984) J. Bacteriol, 158: 1,165, De Louvencourt et al (1983) J. Bacterial, 154: 1,165, Van den Berg et al., (1990) Bio / Technology 8: 135 ); Pichia (Cregg et al (1985) Mol, CeII, Biol 5: 3376, Kunze et al (1985) J. Basic Microbiol 25: 141, US Pat Nos. 4,837,148 and 4,929,555); Saccharomyces (Hinnen et al (1978) Proc Natl Acad Sci USA 75, 1929, Ito et al (1983) J. Bacteriol 153: 163); Schizosaccharomyces (Beach and Nurse (1981) Nature 300: 706); Yarrowia (Davidow et al., (1985) Curr Genet, 10:39, Gaillardin et al (1985) Curr Genet 10:49). The virus used may be, for example, a retrovirus such as Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. An animal according to the present invention is a multicellular living organism, eukaryotic, free of chloroplasts and non-human. In a preferred embodiment, the genetically modified organism used in the present invention is a mammal, preferably a rabbit. Advantageously, the modified human FVII / VIIa of the present invention can be produced in the mammary glands of a mammal, preferably a rabbit, under the control of a specific promoter allowing the expression of said FVII / VIIa in the milk of said mammal. rabbit.

A method for producing a recombinant or transgenic FVII / VIIa in the milk of a transgenic animal may comprise the following steps: a DNA molecule comprising a gene coding for the modified human FVII / VIIa according to the invention, this gene being under the control of a promoter of a protein secreted naturally in milk (such as the casein promoter, beta-casein, lactalbumin, beta-lactoglobulin or the WAP promoter) is integrated into a embryo of a non-human mammal. The embryo is then placed in a female mammal of the same species. Once the mammal from the embryo has grown sufficiently, the lactation of the mammal is induced, then the milk collected. The milk then contains said recombinant or transgenic FVII / VIIa.

An example of a process for the preparation of protein in the milk of a female mammal other than the human is given in EP 0 527 063, the teaching of which may be repeated for the production of the protein of the invention. . A plasmid containing the WAP (Whey Acidic Protein) promoter is manufactured by introducing a sequence comprising the promoter of the WAP gene, this plasmid being made in such a way as to be able to receive a foreign gene placed under the control of the WAP promoter. The plasmid containing the promoter and the gene coding for the protein of the invention are used to obtain transgenic rabbits by microinjection into the male pronucleus of rabbit embryos. Embryos are then transferred in the oviduct of hormonally prepared females. The presence of the transgenes is revealed by the Southern technique from the DNA extracted from the transgenic rabbits obtained. Concentrations in animal milk are evaluated using specific radioimmunoassays. Other documents describe methods for preparing proteins in milk of a female mammal other than man. These include, but are not limited to, US 7,045,676 (transgenic mouse) and EP 1,739,170 (production of von Willebrand factor in a transgenic mammal). These methods of preparation are applicable to the invention using the modified FVII / VIIa DNA according to the present invention.

In a particular embodiment, the genetically modified organism is an insect, for example a mosquito, a fly, etc.

By "recombinant or transgenic FVII / VIIa" is meant any FVII / VIIa obtained from a transformed cell or from a genetically modified organism, that is to say from a microorganism, an animal or a a plant. In contrast, the FVII / VIIa of the invention is not a plasma FVII / VIIa, i.e. it is not a purified product from human or animal plasma.

Thus, the FVII / VIIa of the invention is derived from the transcription and then the translation of a DNA molecule coding for a modified FVII according to the invention and produced by a transgenic cell, a microorganism, a animal or a transgenic plant. . Thus the recombinant or transgenic FVII / VIIa of the invention can be obtained using standard techniques, well known to those skilled in the art, allowing the expression of a protein in a biological system.

The present invention also relates to a method of manufacturing a modified human FVII / VIIa according to the present invention comprising the following steps: a) transforming a cell with a nucleic acid encoding a modified human FVII / VIIa as defined in b) culturing the cell obtained in step a) so that the cell expresses said factor VII / VIIa; and c) purifying the modified human factor VII / VIIa expressed by the transformed cell culture in step b).

The transformed cell is cultured in a suitable medium to express FVII / VIIa. The culture media used are chosen on purpose by the skilled person depending on the cultured cells. Suitable media for cell culture include rIMDM (Iscove's Modified Dulbecco's Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI 1640 or others. These culture media consist mainly of inorganic salts, amino acids, vitamins and other components, including glucose for its energy supply and HEPES for its buffering capacity, basic supplements such as in particular acids amino, minerals, trace elements, specific molecular complements of growth and metabolic activities for each cultured cell type etc.

The present invention also relates to a method for producing a modified human FVII / VIIa according to the present invention in the milk of a transgenic mammal, comprising the steps of: a) obtaining a transgenic mammal which expresses in its mammary glands a nucleic acid coding for a human factor VIII / VIIa modified according to the present invention, b) collecting the milk of the transgenic mammal which contains the factor VII / VIIa, c) purifying the modified human factor VII / VIIa from said collected milk.

Advantageously, the transgenic mammal may be a mouse, a spleen, a rabbit or a goat. Preferably, the transgenic mammal is a rabbit.

The transgenic mammal can be obtained by conventional methods, for example by microinjecting an embryo from a mammal with a DNA sequence coding for a modified human FVII / VIIa according to the present invention, placing said microinjected embryo in the light of the oviduct of a female mammal of the same species, wait for the birth of small mammals from the microinjected embryo, check if the transgenic animal expresses the modified human FVII / VIIa in its milk. The FVII / VIIa of the present invention can be purified by purification methods well known to those skilled in the art, including, but not limited to, chromatography (ion exchange, affinity, hydrophobic, size exclusion), electrophoretic techniques such as preparative isoelectric focusing (IEF), solubility difference (amonium sulphate precipitation) or extraction (Protein Purification J.-C.Janson and Lars Ryden, editors, VCH Publishers, New York (1989)). Preferably, the FVII / VIIa of the invention can be purified by affinity chromatography on a column containing anti-FVII antibodies or on a column containing anti-FVII aptamers. Additional purification can be achieved by conventional techniques of chemical purification, such as HPLC (High Performance Liquid Chromatography). Other purification methods, including barium citrate precipitation, are well known to those skilled in the art and can be used for purification of the FVII / VIIa of the invention.

The term "antibody" as used herein refers to an immunoglobulin or an immunologically active portion thereof, e.g., the antigen-binding region. An antibody therefore refers to a protein comprising at least one, and preferably two, heavy chain and at least one, preferably two light chains.

Aptamer refers to a nucleic acid molecule (DNA or RNA) having a tertiary structure that allows it to specifically bind a protein (Osborne, et al (1997), Curr Opin Chem Biol 1: 5-9; and Patel, DJ (1997) Curr Opin Chem Biol 1: 32-46) Another object of the present invention is a modified human FVII / VIIa composition according to the present invention.

The subject of the present invention is also a pharmaceutical composition containing a modified human FVII / VIIa according to the present invention and an excipient and / or a pharmaceutically acceptable vehicle.

The pharmaceutical composition of the present invention can be used for parenteral, topical or local administration and prophylactically and / or therapeutically. Thus the modified human FVII / VIIa according to the present invention is prepared in a form adapted to the chosen type of administration, for example in liquid form or in freeze-dried form. The modified human FVII / VIIa pharmaceutical compositions according to the present invention may contain an excipient and / or a pharmaceutically acceptable carrier, preferably an aqueous vehicle. Many pharmaceutically acceptable excipients and / or carriers may be used, for example, water, buffered water, saline solution, glycine solution and their derivatives as well as agents necessary to reproduce physiological conditions such as buffering agents and pH adjusters, surfactants such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, this list not being limiting. In addition, the pharmaceutical composition can be sterilized by sterilization techniques well known to those skilled in the art. In general, to manufacture a pharmaceutical composition according to the invention, a person skilled in the art can advantageously refer to the latest edition of the European Pharmacopoeia, for example at the 5th edition of the European Pharmacopoeia published in January 2005, or else again at the 6th edition of the European Pharmacopoeia, available to the public in June 2007. The modified human FVII / VIIa according to the present invention and the pharmaceutical compositions thereof are particularly useful for the manufacture of a medicament. In particular, modified human FVII / VIIa according to the present invention and pharmaceutical compositions thereof are useful for the manufacture of a medicament for the treatment of coagulation disorders in a patient. As disorder of the coagulation treated with a pharmaceutical composition according to the present invention may be mentioned in a non-exhaustive manner multiple bleeding trauma, such as hemophilia A and B or hemorrhages caused by overdosage of anticoagulant.

The modified human FVII / VIIa according to the invention may be used alone or in combination with one or more other pharmaceutically active molecules.

EXAMPLES

Example 1: Establishment of the three-dimensional model of human FVII

The three-dimensional model of human FVII was constructed from an exhaustive study of all the crystallized structures available within the Protein Data Bank (pdb). There are 27 FVII structures that have been analyzed with respect to various parameters such as the expression system, the heavy and light chain integrity, the presence of tissue factor, the resolution, the presence of O- and N-glycosylations. , the presence of γ-carboxylation and the date of publication at the protein data bank (pdb). From this study, the protein structure was constructed after corrections, assembly and minimization of structures. The software suite used is Sybyl v7.2 (Tripos, Inc.). Sybyl is a modeling software based on the minimization of the total energy thus making it possible to define the most stable structure and thus the most probable. The overall minimization step comprising fixing the "BackBone" of the protein by simulating the presence of tissue factor, has been performed under the following conditions:

- stop parameter: energy gradient <0.5 kCal / mol or maximum number of iterations reached = 10000

- minimization method: Powell - force field: Amber7FF99

- method of calculating the charges for the glycoprotein: Amber7FF99,

- load calculation method for ions and active site inhibitor: Gasteiger-Hϋckel

- non-bonded cut-off: 8 A.

EXAMPLE 2 Extraction and Purification of FVII Obtained in Transgenic Rabbit Milk a) Extraction of FVII

A volume of 500 ml of raw non-skimmed milk was diluted with 9 volumes of 0.25 M sodium phosphate buffer, pH 8.2. After stirring for 30 minutes at room temperature, the aqueous phase enriched with FVII was centrifuged at 10,000 g for 1 hour at 15 ° C. (Sorvall Evolution RC centrifuge - 6700 rpm - rotor

SLC-6000). 6 pots of about 835 ml are needed.

After centrifugation, three phases are present: a surface lipid phase (cream), a clear non-lipidic aqueous phase enriched in FVII (majority phase) and a solid white phase in pellet (precipitates of insoluble caseins and calcium compounds).

The non-lipidic aqueous phase FVII was collected at the peristaltic pump until the creamy phase. The creamy phase was collected separately. The solid phase (precipitate) was removed.

The non-lipidic aqueous phase, however still comprising very small amounts of lipids, was filtered on a filter sequence (PaII SLK7002U010ZP - 1 μm pore size glass fiber pre-filter - then PaII SLK7002NXP - nylon 66 0.45 μm pores). At the end of filtration, the lipid phase was passed on this filtration sequence which completely retains the lipid globules of the milk, and the filtrate is clear.

The filtered non-lipidic aqueous phase was then dialyzed on an ultrafiltration membrane (Millipore Biomax 50 kDa - 0.1 m ² ) to make it compatible with the chromatography phase. The molecular weight FVII of about 50 kDa does not filter through the membrane, unlike the milk salts, sugars and peptides. Initially, the solution (about 5000 ml) is concentrated to 500 ml, and then dialysis by ultrafiltration while maintaining the constant volume allows to remove the electrolytes and condition the biological material for the chromatography step. The dialysis buffer is a 0.025M sodium phosphate buffer, pH 8.2.

This non-lipidic aqueous phase comprising FVII can be assimilated to whey enriched in FVII-tg. This preparation is stored at -30 ° C before continuing the process.

The non-lipidic aqueous phase comprising FVII at the end of this step is perfectly clear and is compatible with the chromatographic steps that follow.

Approximately 93,000 IU of fVII-tg are extracted at this stage. The purity in FVII of this preparation is of the order of 0.2%.

b) Purification of the FVII

1. Chromatography on hydroxyapatite gel

An Amicon 90 column (9 cm diameter - 64 cm ² section) was filled with BioRad Ceramic Hydroxyapatite Gel Type I (CHT-I).

The gel was equilibrated in buffer A consisting of a mixture of 0.025 M sodium phosphate and 0.04 M sodium chloride, pH 8.0. The entire preparation stored at -30 ° C is thawed in a water bath at 37 ° C until complete dissolution of the ice cube and then was injected on the gel (linear flow 100 cm / h, or 105 ml / min). The unbound fraction was removed by passing a buffer consisting of 0.025 M sodium phosphate and 0.04 M sodium chloride, pH 8.2, until baseline return (RLB).

Elution of the fraction containing FVII was made by buffer B consisting of 0.25 M sodium phosphate and 0.4 M sodium chloride, pH 8.0. The eluted fraction was collected until the return to baseline.

This chromatography makes it possible to recover more than 90% of the FVII, while eliminating more than 95% of the lactic proteins. The specific activity (AS) is multiplied by 25. About 85,000 IU of FVII of 4% purity are available at this stage. 2. 100 kDa tanαential filtration and 50 kDa concentration / dialysis The entire eluate from the previous step was filtered in tangential mode on a 100 kDa ultrafiltration membrane (PaII OMEGA SC 100K - 0.1 m ² ). FVII has been filtered through the 100 kDa membrane, whereas proteins with a molecular weight greater than 100 kDa are not filterable.

The filtered fraction was then concentrated to about 500 ml and dialyzed on the 50 kDa ultrafilter already described in Example 1. The dialysis buffer was 0.15 M sodium chloride.

At this stage of the process, the product is stored at -30 ° C. before passing through ion exchange chromatography.

This step made it possible to reduce the protein load of molecular weight greater than 100 kDa and in particular proenzymes. The 100 kDa membrane treatment retains about 50% of proteins including high molecular weight proteins, while filtering 95% of FVII, or 82,000 IU of FVII. This treatment makes it possible to reduce the risks of proteolytic hydrolysis during the downstream stages.

3.Q-Sepharose® FF gel chromatograms

These three successive chromatographies on Q-Sepharose® Fast Flow (QSFF) ion exchange gel were carried out to purify the active principle, to allow activation of FVII in activated FVII (FVIIa) and finally to concentrate and formulate the FVII composition.

3.1 Step Q-Sepharose® FF 1 - "High Calcium" elution A column 2.6 cm in diameter (5.3 cm ² in section) was filled with 100 ml of Q-Sepharose® FF gel (GE Healthcare).

The gel was equilibrated in 0.05 M Tris buffer, pH 7.5.

The entire fraction stored at -30 ° C. was thawed in a water bath at

37 ° C until complete dissolution of the ice cube. The fraction was diluted at ^λ h [v / v] with the equilibration buffer before injection on the gel (flow rate 13 ml / min, ie linear flow of

150 cm / h), then the unbound fraction was removed by buffer

RLB.

A first low FVII protein fraction was eluted at 9 ml / min (100 cm / hr) with a buffer of 0.05 M Tris and 0.15 M sodium chloride, pH 7.5, and was then eliminated.

A second protein fraction rich in FVII was eluted at 9 ml / min (ie 100 cm / h) with a buffer of 0.05 M Tris, 0.05 M sodium chloride and 0.05 M calcium chloride. pH 7.5.

This second fraction was dialyzed on the 50 kDa ultrafilter already described in Example 1. The dialysis buffer is 0.15 M sodium chloride. was stored at + 4 ° C overnight before the ^2θmθ pass through anion exchange chromatography. This step makes it possible to recover 73% of the FVII (ie 60000 IU of FVII), while eliminating 80% of the accompanying proteins. It also allows the activation of FVII in FVIIa.

3.2 Step Q-Sepharose® FF 2 - elution "Low Calcium"

A 2.5 cm diameter column (4.9 cm ² section) was filled with 30 ml of Q-Sepharose® FF gel (GE Healthcare).

The gel was equilibrated in 0.05 M Tris buffer, pH 7.5. The previous eluted fraction (second fraction), stored at + 4 ° C., was diluted before injection on the gel (flow rate 9 ml / min, ie linear flow rate of 100 cm / h).

A fraction containing very high purity FVII was eluted at 4.5 ml / min (ie 50 cm / h) in buffer of 0.05 M Tris, 0.05 M sodium chloride and 0.005 M calcium chloride. pH 7.5. Approximately 23,000 IU of FVII were purified, ie, 12 mg of FVII.

This step eliminates more than 95% of the accompanying proteins (rabbit milk proteins).

This eluate, of greater than 90% purity, has structural and functional characteristics close to that of native human FVII. It was concentrated and formulated by the third pass in ion exchange chromatography.

3.3 Step Q-Sepharose® FF 3 - elution "Sodium"

A 2.5 cm diameter column (4.9 cm ² section) was filled with 10 ml of Q-Sepharose® FF gel (GE Healthcare). The gel was equilibrated in 0.05 M Tris buffer, pH 7.5.

The purified eluted fraction of the previous step was diluted five times with purified water for injection (PPI) before injection on the gel (flow rate 4.5 ml / min, ie linear flow of 50 cm / h).

FVII was then eluted at a rate of 3 ml / min (36 cm / h) with 0.02 M Tris buffer and 0.28 M sodium chloride, pH 7.0.

A composition of FVII in the form of a concentrate was prepared with purity greater than 95%. The product is compatible with intravenous injection. The process has a cumulative yield of 22%, which makes it possible to purify at least 20 mg of FVII per liter of milk used. FVII can then be subjected to different structural analyzes, as developed in the examples that follow. Example 3 Identification of Atypical Cleavages of FVII by MALDI-TOFMS

MALDI-TOF MS (Matrix-Assisted Laser Desorption / lonization Time of Flight Mass Spectrometry) is a technique for measuring the molecular mass of molecules with high precision. The proteins to be analyzed were mixed with a matrix that absorbs at the wavelength of the laser used. The main matrices are D-cyano-4-hydroxycinnamic acid (HCCA) for peptide analysis, sinapinic acid (SA) for proteins and 2,5-dihydroxybenzoic acid (DHB) for oligosaccharides.

The method involves irradiating matrix / analyte co-chstals with a laser, resulting in joint desorption of the template and analyte molecules. After ionization in the gas phase, the analyte molecules reach a time of flight detector. Mass and flight time being directly related, the measurement of the latter allows the determination of the mass of the analyte. The identification of each protein or peptide can be carried out by measuring its mass observed in mass spectrometry, compared with the theoretical mass deduced from the FVII sequence. The instrument used is a Bruker Autoflex II operating in the TOF and TOF / TOF modes.

The MALDI-TOF spectrum of FVII shows a 14.7 kDa form (FIG. 1, polypeptide IV) which corresponds to the C-terminal peptide [Gly ₂ 9i-Pro ₄₀ 6] of the heavy chain containing Asn ₃ 22 glycosylated predominantly by a oligosaccharide of monosialylated biantenene type (A1) and other glycans (A1 F, A2, ...). The presence of the complementary form (FIG. 1, polypeptide IV), N terminal of FVII, at 34.6 kDa ending with arginine 290 is also observed on the spectrum. Another atypical cleavage is also present at 44.8 kDa (FIG. polypeptide II) which corresponds to the cleavage of the light chain after lysine 38, that is to say to a form of FVII amputated from the GIa domain and thus whose affinity for tissue factor is decreased.

Under reducing conditions (FIG. 2), the presence of the heavy and light chains of FVIIa at 29.9 and 19.3 kDa (polypeptide I), respectively, is noted. Another form is observed at 1.9 kDa corresponding to the [LyS ₃ -I ₆ -Pro ₄₀ 6] peptide containing glycosylated R 2 ₃₂ . In the native state, this peptide is bound to the N-terminal portion of the protein by a disulfide bridge (Cys ₃ io-Cys ₃ 29).

All analyzed FVII samples have one or more of these truncated forms. All of the forms identified result from "serine protease" type cuts. These different cleavages can therefore be of autocatalytic origin.

Example 4 Quantification of Atypical Cleavages by Sequencing of Edman

N-terminal sequencing of FVII was performed on a microsequencer (Procise

491 HT; Applied Biosystem) according to the principle of chemical degradation of Edman which consists of three steps: coupling - cleavage and conversion and separation of amino acids generated on reverse phase column. The N-terminal amino acids thus generated are then analyzed and identified by means of amino acids standards and compared to the theoretical sequence of the protein studied. The results were exploited after data acquisition and analysis by comparison with a standard amino acid chromatogram (SequencePro Applied Biosystems). The analyzed FVII sequence was compared to the theoretical amino acid sequence.

2 majority sequences have been systematically identified:

N-terminal sequence LC: ANAFLEELRPGSLERECKEEQCSF (SEQ ID NO: 3)

- N terminal sequence HC: IVGGKVCPKGECPWQVLLLVNGAQLCG (SEQ ID No. 4)

3 other sequences, present according to the products, were identified:

LC sequence: LFWISYSDGDQ (SEQ ID No. 5) (atypical cleavage after lysine 38).

HC sequence: ATALELM VLN VP RLMTQ (SEQ ID NO: 6) (atypical cleavage after arginine 290).

HC sequence: KVGDSPΛ / ITEYMFCAGYSDGS (SEQ ID No. 7) (atypical cleavage after arginine 315).

The amino acids represented in bold and italic indicate sequence holes, that is to say unidentified amino acids in Edman sequencing because of the presence of post-translational modifications such as D-carboxylation, N- or O -glycosylation. A quantitative evaluation was performed to estimate the proportion of different atypical cuts as a function of FVII. The results are shown in Table 1 below:

Table 1. Percentages of different atypical cuts compared to the total product, based on the origins of FVII. FVII-pd: human plasma FVII;

FVII-Tg: transgenic non-mutated human FVII; FVII-rec: non-mutated commercial recombinant human FVII

The light chain of FVII has an atypical cleavage rate between amino acids K38 and L39 varying between 4.5 and 26% depending on the origin of the product. The heavy chain of FVII has an atypical cleavage between R315 and K316 (varying between 9 and 52% depending on the origin of the product) and is also cleaved between R290 and G291 (varying between 4 and 13% depending on the origin of the product).

Claims

1. Human factor VII / VIIa modified with respect to the native human factor VII / VIIa peptide sequence, wherein at least two amino acids selected from lysine 38, arginine 290 and arginine 315 are substituted or deleted, characterized in that:

said lysine 38 is substituted with an amino acid selected from glutamine, alanine, glutamic acid, glycine, isoleucine, leucine, methionine, histidine, phenylalanine, praline, serine, threonine tryptophan, tyrosine or valine;

said arginine 290 is substituted with an amino acid chosen from glutamine, alanine, glutamic acid, asparagine, glycine, isoleucine, leucine, methionine, histidine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine or valine and / or

said arginine 315 is substituted with an amino acid chosen from glutamine, alanine, glutamic acid, asparagine, glycine, isoleucine, leucine, methionine, histidine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine or valine.

2. The modified human factor VII / VIIa according to claim 1, characterized in that the lysine 38 is substituted by an amino acid selected from glutamine, histidine or glutamic acid.

3. The modified human factor VII / VIIa according to claim 1, wherein arginine 290 is substituted with an amino acid selected from glutamine, histidine, asparagine or glutamic acid. .

4. modified human factor VII / VIIa according to any one of claims 1 to 3, characterized in that the arginine 315 is substituted by an amino acid selected from glutamine, histidine, asparagine or glutamic acid .

5. Modified human factor VII / VIIa according to any one of claims 1 to 4, characterized in that lysine 38 is substituted with glutamine.

6. Human factor VII / VIIa modified according to any one of claims 1 to

5, characterized in that arginine 290 is substituted with glutamine.

7. The modified human factor VII / VIIa according to any one of claims 1 to

6, characterized in that arginine 315 is substituted with glutamine.

Nucleic acid encoding a modified human VII / VIIa factor defined by any one of claims 1 to 7.

An expression vector in which a nucleic acid is inserted according to claim 8.

The nucleic acid transformed cell of claim 8 expressing a modified human VII / VIIa factor.

A genetically modified non-human organism comprising in its genome a nucleic acid encoding a modified human factor VII / VIIa according to any one of claims 1 to 7, and expressing said modified human factor VII / VIIa.

12. Genetically modified organism according to claim 1 1, characterized in that said genetically modified organism is a microorganism, an animal or a plant.

13. Genetically modified organism according to one of claims 1 1 or 12, characterized in that said genetically modified organism is a mammal.

14. Genetically modified organism according to claim 13, characterized in that said mammal is a rabbit.

15. Genetically modified organism according to claim 1 1 or 12, characterized in that said genetically modified organism is an insect.

16. A method of manufacturing a factor VII / VIIa defined by any one of claims 1 to 7 comprising the following steps: a) in vitro transformation of a cell with a nucleic acid encoding a

Modified human FVII / VIIa according to any one of claims 1 to 7, b) culturing the cell obtained in step a) so that the cell expresses said factor VIII / VIIa, and c) purifying the modified human factor VII / VIIa expressed by the transformed cell cultured in step b).

A method of manufacturing a modified human VII / VIIa factor according to any one of claims 1 to 7 in the milk of a transgenic non-human mammal, comprising the steps of: a) obtaining a transgenic mammal that expresses in its mammary glands, a nucleic acid encoding a human factor VII / VIIa modified according to one of the claims 1 to 7, b) collecting the milk of the transgenic mammal that contains the factor VII / VIIa, and (c) purifying the modified human factor VII / VIIa. from said collected milk.

18. The manufacturing method according to claim 17, characterized in that the transgenic animal is selected from a mouse, a spleen, a goat and a rabbit.

19. The manufacturing method according to claim 18, characterized in that the transgenic animal is a rabbit.

20. Factor VIII / VIIa composition, characterized in that said composition contains a modified human VII / VIIa factor defined by any one of Claims 1 to 7.

21. A pharmaceutical composition comprising a modified human VII / VIIa factor as defined by any one of claims 1 to 7, and a pharmaceutically acceptable excipient and / or carrier.

22. Use of a factor VII / VIIa according to any one of claims 1 to 7 for the manufacture of a medicament.

23. Use of a factor VIII / VIIa according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of coagulation disorders.

24. Use of a factor VII / VIIa according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of multiple bleeding trauma.

25. Use of a factor VII / VIIa of any one of claims 1 to 7 for the manufacture of a medicament for the treatment of hemophilia.

26. Use of a factor VIII / VIIa of any one of claims 1 to 7 for the manufacture of a medicament for the treatment of hemorrhages caused by an overdose of anticoagulant.