WO2017050820A1 - Protéines de fusion du facteur viii - Google Patents

Protéines de fusion du facteur viii Download PDF

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Publication number
WO2017050820A1
WO2017050820A1 PCT/EP2016/072414 EP2016072414W WO2017050820A1 WO 2017050820 A1 WO2017050820 A1 WO 2017050820A1 EP 2016072414 W EP2016072414 W EP 2016072414W WO 2017050820 A1 WO2017050820 A1 WO 2017050820A1
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fviii
seq
domain
fusion
amino acids
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PCT/EP2016/072414
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Gert Bolt
Marianne Hjortnaes Kjalke
Ditte Maria Karpf
Jakob Ewald Rasmussen
Lars Thim
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Novo Nordisk A/S
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Publication of WO2017050820A1 publication Critical patent/WO2017050820A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to treatment and/or prophylaxis of haemophilia using FVIII fusion proteins.
  • Haemophilia A is an inherited bleeding disorder wherein formation of the blood clot in the patients occurs normally but the clot is unstable due to a lack of secondary thrombin formation.
  • the disease is treated by intravenous injection of coagulation factor VIII (FVIII) isolated from blood or produced recombinantly.
  • FVIII coagulation factor VIII
  • the circulatory in vivo half life of endogenous FVIII is 12-14 hours and prophylactic treatment with FVIII is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients.
  • Recombinant FVIII variants with a prolonged in vivo circulatory half life e.g.
  • heterologous FVIII:Fc fusions are becoming available for the haemophilia patients.
  • Another frequently used strategy for prolonging FVIII circulatory half life is conjugation of FVIII, employing chemical or enzymatic methods.
  • a therapies offering compounds with significantly prolonged circulatory half life that are preferably relatively simple to produce on an industrial scale.
  • the present invention relates to FVIII fusion proteins comprising a FVIII molecule and a fusion partner, wherein said fusion partner is inserted at the FVIII light chain N- terminus at position D1658, wherein said fusion partner thus replaces amino acids E1649- S1657 according to SEQ ID NO 1. Amino acids E1649-S1657 are thus absent from the FVIII fusion proteins according to the invention.
  • the present invention furthermore relates to pharmaceutical compositions suitable for treatment of blood clotting diseases.
  • treatment refers to the medical therapy of any human or other vertebrate subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to treating a disease in said human or other vertebrate.
  • the timing and purpose of said treatment may vary from one individual to another, according to the subject's health.
  • said treatment may be prophylactic, palliative, symptomatic and/or curative.
  • Compounds and pharmaceutical compositions according to the invention may be administered parenterally, such as e.g. intravenously or extravascularly (such as e.g. intradermally, intramuscularly, subcutaneously, etc). Compounds and pharmaceutical compositions according to the invention may be administered
  • Factor VIII is a large, complex glycoprotein that is primarily produced by endothelial cells including liver sinusoidal endothelial cells (LSECs) and possibly also hepatocytes.
  • Human FVIII codes for 2351 amino acids, including a signal peptide, and contains several distinct domains as defined by homology. There are three A- domains, a unique B-domain, and two C-domains. The domain order can be listed as NH2- A1 -A2-B-A3-C1-C2-COOH.
  • A1 -a1- A2-a2-B chain is termed the heavy chain (HC) while the a3-A3-C1 -C2 is termed the light chain (LC).
  • the chains are connected by bivalent metal ion-bindings.
  • Table a FVIII domains and regions. The numbering of domains, regions and single amino acid residues in the Factor VIII molecule follow the numbering of full length Factor VIII (also if the B-domain is truncated or if a fusion partner is added to the molecule).
  • the nucleotide sequence encoding full length Factor VIII encodes a B domain of 908 amino acid residues.
  • the B-domain in full-length FVIII is processed, resulting in a mixture of heavy chain with different length of B-domains attached (Jankowski MA et al. Haemophilia 2007; 13: 30-37).
  • rFVIII with truncated B domains may comprise B domains being significantly shorter than 908 amino acids - one example of a truncated B domain is the 21 amino acid B domain linker according to SEQ ID NO 2.
  • FVIII variants comprise an a3 region spanning amino acids 1655-1689 and 1658-1689 (Lind P et al. Eur J Biochem 1995; 232: 19-27). Such FVIII proteins, as well as other naturally occurring FVIII variants, are also part of the present invention.
  • Endogenous FVIII molecules circulate in vivo as a pool of molecules with B domains of various sizes, the shortest having C-terminal at position 740, i.e. at the C-terminal of A2- a2, and thus contains no B domain.
  • FVIII molecules with B-domains of different length all maintain procoagulant activity.
  • FVIII Upon activation with thrombin, FVIII is cleaved C-terminal of A1 -a1 at position 372, C-terminal of A2-a2 at position 740, and between a3 and A3 at position 1689, the latter cleavage releasing the a3 region with concomitant loss of affinity for VWF.
  • the activated FVIII molecule is termed FVIIIa.
  • the activation allows interaction of FVIIIa with phospholipid surfaces like activated platelets and activated factor IX (FIXa), i.e. the tenase complex is formed, allowing efficient activation of factor X (FX) resulting in thrombin generation and ultimately formation of a fibrin-stabilized haemostatic clot.
  • FIXa activated factor IX
  • FX factor X
  • Wildtype(wt)/native FVIII is the human FVIII molecule derived from the full length sequence as shown in SEQ ID NO: 1 (amino acid 1 -2332).
  • FVIII includes natural allelic variants of FVIII with FVIII activity. It follows that FVIII fusion proteins according to the present invention are recombinantly produced proteins (rFVIII), using well known methods of production and purification. The degree and location of glycosylation, tyrosine sulfation and other post-translation modifications of FVIII occurring in the cell may therefore vary, depending on the chosen host cell and its growth conditions.
  • FVIII fusion proteins herein are capable of functioning in the coagulation cascade in a manner that - on a molar basis - is functionally similar, or equivalent, to wt/endogenous FVIII, inducing the formation of FXa via interaction with FIXa on an activated platelet and supporting the formation of a blood clot.
  • FVIII(a) activity can be assessed in vitro using techniques well known in the art. Clot analyses, FX activation assays (often termed chromogenic assays), thrombin generation assays and whole blood thrombo- elastography are examples of such in vitro techniques.
  • FVIII molecules according to the present invention have FVIII activity that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100% or even more than 100% of that of native human FVIII.
  • Native human FVIII is herein understood to be either plasma derived purified FVIII or recombinant FVIII corresponding closely to SEQ ID NO 1 , or a B domain truncated version thereof.
  • Endogenous full length FVIII is synthesized as a single-chain precursor molecule. Prior to secretion, the precursor is cleaved into the heavy chain and the light chain.
  • Recombinant FVIII can be produced by means of two different strategies. Either the heavy chain with or without the B-domain and the light chain are synthesized individually as two different polypeptide chains (two-chain strategy) or the B domain-truncated FVIII is synthesized as a single precursor polypeptide chain (single-chain strategy) that is cleaved into the heavy and light chains in the same way as the full-length FVIII precursor.
  • a B domain-truncated FVIII (precursor) fusion polypeptide according to the invention produced by the single-chain strategy, the heavy and light chain moieties are often separated by a linker.
  • the sequence of the linker is preferably derived from the FVIII B-domain.
  • the C- terminal part of the linker contains a furin recognition site resulting in intracellular processing into a heavy and a light chain.
  • amino acid 1644-1648 constitutes this recognition site.
  • the thrombin cleavage site leading to removal of the linker during activation of B domain-deleted FVIII is located in the a2 region C-terminal to the A2 domain.
  • the size and amino acid sequence of the linker is unlikely to influence its removal from the remaining FVIII molecule by thrombin activation. Truncation of the B domain is often considered to be an advantage for recombinant production of FVIII.
  • parts of the B domain can be included in the B domain linker herein without reducing the productivity.
  • SEQ ID NO: 1 wt human FVIII (Ser750 residue shown in bold and underline) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTWITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV
  • B domain truncated and B domain deleted FVIII are used interchangeably herein.
  • the B domain in FVIII spans amino acids 741-1648 of SEQ ID NO: 1.
  • the B domain undergoes endo-proteolysis at several different sites, generating large heterogeneity in circulating plasma FVIII molecules as explained above and in Jankowski et al, Haemophilia 2007; 13: 30-37 and D'Amici et al, Electrophoresis 2010; 31 : 2730-2739 . While the B-domain plays a role in intracellular expression of FVIII, the exact extracellular function of the heavily glycosylated B domain, if any, is unknown.
  • the B domain is dispensable for FVIII activity in the coagulation cascade. Recombinant FVIII is thus frequently produced in the form of B domain-truncated variants.
  • the FVIII fusion protein is produced by an expression vector encoding a FVIII molecule comprising a 21 amino acid residue linker (B domain linker) sequence with the following sequence: SEQ ID NO 2: SFSQNSRHPSQNPPVLKRHQR.
  • An O-glycan is attached to the underlined S in SEQ ID NO 2 - this residue corresponds to position S750 in SEQ ID N01 .
  • the FVIII fusion protein herein comprises a linker sequence with the following sequence: SEQ ID NO: 3: SFSQNSRHPSQNPPVLKRHQ.
  • the FVIII fusion protein herein comprises a linker sequence with the following sequence: SEQ ID NO: 4: FSQNSRHPSQNPPVLKRHQR.
  • the FVIII fusion protein herein are B domain truncated FVIII variants comprising an O-glycan attached to the Ser 750 residue shown in SEQ ID NO 1 - the B domain linker sequence may in that case optionally be selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.
  • FVIII fusion proteins herein may be FVIII molecules comprising the full B domain, or it may be comprise a FVIII molecule comprising a truncated B domain - the FVIII molecule herein furthermore has FVIII activity.
  • FVIII molecules according to the invention comprise a B domain of 908, 4-908, 10-908, 15-908, 4-900, 5-900, 10-900, 15-900, 20-900, 25-900, 50- 900, 100-900, 200-900, 300-900, 400-900, 500-900, 600-900, 700-900, 10-700, 15-700, 20- 700, 25-700, 50-700, 100-700, 200-700, 300-700, 400-700, 500-700, 600-700, 10-500, 15- 500, 20-500, 25-500, 50-500, 100-500, 200-500, 300-500, 400-500, 10-400, 15-400, 20-400, 25-400, 50-400, 100-400, 200-400, 300-400, 10-
  • the inventors of the present invention have made the surprising observation that the FVIII fusion proteins herein results in a FVIII fusion protein with a better yield and/or more simple production process, and/or improved therapeutic properties.
  • the FVIII part of this fusion protein may be a B domain truncated FVIII molecule comprising a B domain linker with an amino acid sequence selected from SEQ ID No 2, 3, or 4.
  • the FVIII part of the protein may alternatively be a full length FVIII protein comprising the entire B domain.
  • Fusion proteins are proteins created through the in-frame joining of two or more DNA sequences which originally encoded FVIII and the FVIII fusion partner. Translation of the fusion protein DNA sequence will result in a single protein sequence which may have functional properties derived from each of the original proteins or peptides.
  • DNA sequences encoding fusion proteins may be created by standard molecular biology methods such as overlapping PCR or DNA ligation - the FVIII fusion partner according to the present invention is preferably inserted at the FVIII light chain N-terminus at position D1658 - and thus replacing positions E1649-S1657 (according to the sequence in SEQ ID NO 1 - amino acid positions according to SEQ ID NO 1 ).
  • the resulting fusion protein DNA sequence may be inserted into an appropriate expression vector that supports fusion protein expression in a standard host organism.
  • Fusion proteins may furthermore contain a linker or spacer peptide sequence that separates the protein or peptide parts which define the fusion protein.
  • the linker or spacer peptide sequence may facilitate the correct folding of the individual protein or peptide parts and may make it more likely for the individual protein or peptide parts to retain their individual functional properties.
  • Linker or spacer peptide sequences may be inserted into fusion protein DNA sequences during the in frame assembly of the individual DNA fragments that make up the complete fusion protein DNA sequence i.e. during overlapping PCR or DNA ligation.
  • linker sequences according to the invention comprising repetitive GS residues include e.g.: (GS) n , (GGGS) n , (GGGGS) n , (GGGS) n (GGG), etc.
  • the linker can thus be very short, e.g. 1 , 2, 3, 4, 5, 6, 7, or 8 amino acids or somewhat longer, e.g. 5-10, 5-15, 5-20, 5- 30, 5-50, 10-15, 10-20, 10-30, 10-40, 10-50, 15-20, 15-30, 15-40, 15-50 amino acids.
  • the fusion proteins according to the present invention are FVIII proteins fused to a fusion partner.
  • the fusion partner (or optionally a linker sequence bridging FVIII and the fusion partner) is fused to the FVIII light chain terminus at position D1658 - thus replacing/deleting residues E1649-S1657 from the FVIII molecule (relative to the amino acid sequence according to SEQ ID NO 1 ).
  • the fusion partner may alternatively be fused to position Q1659, E1660, E1661 , 11662, or even position D 1663 relative to the amino acid sequence according to SEQ ID NO 1 .
  • FVIII DNA sequence encodes amino acid residue E1649-R1689 (according to the sequence set forth in SEQ ID NO 1 )
  • a significant fraction - up to about 90% depending on production method - of FVIII protein has an N-terminus at position D1658 - likely due to endo-proteolytical processing of the S1657-D1658 sequence in the production cell. It is possible that processing at the N-terminal FVIII light chain is done by a metallo-protease - in which case it may also take place at one of the D1658 neighbouring amino acid residues. In connection with the present invention, this protease site is thus presumed to be removed by deletion.
  • an alternative solution to the light chain endo-proteolytical problem may be to introduce mutations in the FVIII light chain N-terminus in order to remove the protease site - a preferred solution is, however, to remove the protease site by deletion.
  • the inventors have herein shown that it is possible to solve these problems by fusing the fusion partner (or the linker attached to the fusion partner) e.g. at position D1658 (according to the sequence in SEQ ID NO 1 ). Deletion of amino acids E1649-S1657 is not associated with any negative impact - the resulting molecules are safe in use, have FVIII activity, are produced with good yields, etc. Furthermore, position Y1664 and the acidic amino acid residues surrounding Y1664 are maintained.
  • FVIII B domain fusion partner A "FVIII B domain fusion partner" according to the present invention is derived from the FVIII B domain.
  • the only modification compared to the wt FVIII B domain is that the FVIII B domain fusion partner may be truncated, i.e. a region has been removed/deleted relative to the wt FVIII B domain.
  • two, three or more truncations were made in the FVIII B domain fusion partner
  • minor amino acid modifications (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 point mutations in the form of amino acid alterations, -additions and/or -deletions) may be present in the FVIII B domain fusion partner
  • the FVIII fusion proteins fused to a FVIII B domain fusion partner according to the present invention are thus structurally similar to the native FVIII molecule in that the fusion partner is derived from the FVIII B domain rather than being derived from a heterologous fusion molecule.
  • FVIII fusion proteins fused to a FVIII B domain fusion partner can thus be said to be a homologous type of FVIII fusion protein with a desirable safety profile.
  • the FVIII B domain fusion partner/B domain fusion partner according to the present invention can be regarded as an "additional FVIII B domain" having a size of 100-908 amino acids.
  • the size of the FVIII B domain fusion partner is 100-900, 100-800, 100- 700, 100-600, 100-500, 100-400 amino acids, preferably 150-650, more preferably 150-600, more preferably 150-550, more preferably 150-500, more preferably 150-450, more preferably 150-400, more preferably 150-350, more preferably 200-700, more preferably 200-600, more preferably 200-500, more preferably 200-400, more preferably 200-300, and most preferably about 200 to 250 amino acids.
  • the FVIII fusion proteins according to the invention may even comprise more than one FVIII B domain fusion partners, such as e.g. two, three, or even four FVIII B domain fusion partners fused head-to-tail to the FVIII light chain N-terminus as described herein and even fused to or within different domains of the FVIII molecule.
  • FVIII fusion molecules according to the present invention may thus comprise a FVIII molecule fused to two, three, or four identical or different FVIII B domain fusion partners as defined herein.
  • the FVIII in vivo circulatory half life can be increased by fusing a FVIII derived B domain fusion partner to a FVIII molecule. This effect may be caused by an ability of this additional FVIII B domain(-s) to interfere with various FVIII clearance receptors.
  • An explanation of the apparent ability of FVIII B domain fusion partners to interfere with FVIII clearance may be that the FVIII B domain tends to fold in a less structured and compact way compared to other proteins - thus mimicking the effect of large bulky groups frequently attached to therapeutic proteins to prolong the half life thereof, such as e.g. polymeric groups (e.g.
  • FVIII B domain fusion partners may have a relatively low tendency to form secondary and/or tertiary protein structures such as e.g. alpha helix, beta sheets and folded domains. Secondary structures can be measured by e.g. circular dichroism (CD).
  • CD circular dichroism
  • Another feature of the FVIII B domain is a tendency to be heavily glycosylated with N-glycans but likely also several O-glycans - this structure may provide the B domain with a large hydrodynamic volume in a similar manner as e.g. PEG polymers, polysaccharide polymers, etc.
  • FVIII B domain fusion partners according to the present invention may furthermore have the advantage of being relatively homogenous in structure - in the production cell line as well as in in vivo circulation.
  • An example thereof is the 226 amino acid B domain fusion partner corresponding to SEQ ID NO 1 1 - this FVIII B domain fusion partner tends to avoid endo-proteolytical processing. The tendency to undergo this processing can be analysed and quantified using established techniques such as e.g. HPLC, SDS-PAGE, etc.
  • B domain fusion partners include: SEQ ID no. 5 (FVIII amino acids 741 -769):
  • SEQ ID no. 7 (FVIII amino acids 741 -857):
  • SEQ ID no. 8 (FVIII amino acids 741 -903):
  • SEQ ID no. 12 (FVIII amino acids 741 -968):
  • SEQ ID no. 13 (FVIII amino acids 741 -1003):
  • SEQ ID no. 14 (FVIII amino acids 741 -1009):
  • SEQ ID no. 17 (FVIII amino acids 741 -1070):
  • SEQ ID no. 26 (FVIII amino acids 741 -1444):
  • SEQ ID no. 30 (FVIII amino acids 741 -1524):
  • IgG Fc domain will often be preferred due to the relatively long circulatory half-life of IgG antibodies.
  • the Fc domain may furthermore be modified in order to modulate certain effector functions such as e.g. complement binding and/or binding to certain Fc receptors.
  • Fusion of FVIII with an Fc domain, which has the capacity to bind to FcRn receptors, will generally result in a prolonged in vivo circulatory half-life. Mutations in positions 234, 235 and 237 in an IgG Fc domain will generally result in reduced binding to the FcyRI receptor and possibly also the FcyRlla and the FcyRIII receptors.
  • a modified IgG Fc domain of a fusion protein according to the invention comprises one or more of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1 q-mediated complement fixation (A330S and P331 S), respectively.
  • the Fc domain may be an lgG4 Fc domain, preferably comprising the S241 P/S228P mutation.
  • the fusion partner can be an Fc receptor that assumingly can extend the FVIII half life by interaction with immunoglobulins.
  • Fc receptors are cell surface receptors that recognize and bind the Fc portion of antibodies. Based on structure, cell distribution and affinity to IgG, the Fc receptors are divided into three classes: FcyRI (CD64), FcvRII (CD32), and FcvRII I (CD16).
  • Fc receptors bind to antibodies that are attached to infected cells or invading pathogens.
  • the Fc receptor can be FcyRI (CD64 -Cluster of Differentiation 64) or a portion thereof, e.g. the extracellular portion.
  • the Fc receptor can be FcyRII or FcyRIII, or a portion thereof, e.g. the extracellular portion.
  • the FVIII fusion protein according to the invention comprises a
  • FVIII molecule fused to albumin.
  • FVIII-albumin fusion proteins delay in vivo clearance of FVIII by interaction/binding with the FcRn receptor.
  • FVIII is fused to a fusion partner to delay in vivo clearance of FVIII by interaction with platelets.
  • a fusion partner includes single-chain (SC) antibodies as well as Fab fragments of antibodies binding to proteins on the platelet surface such as e.g. GPIIIa SC antibodies.
  • FVIII can be fused to a fusion partner to delay in vivo clearance of FVIII by shielding.
  • a non-limiting example thereof includes polypeptides with stretches of non-hydrophobic amino acids ("unstructured polypeptides") such as Sequence A (seq A - a non-limiting example is set forth in SEQ ID NO 39).
  • FVIII can be fused to a fusion partner to delay in vivo clearance of FVIII by unknown mechanisms.
  • a fusion partner includes: growth hormone binding protein (GHBP), parts of vWF, vWF binding protein, and parts of chorion gonadotropin.
  • FVIII can be fused to a fragment derived from VWF: VWF(amino acids 764-828), VWF(amino acids 764-865), VWF(amino acids 764-1035), VWF(amino acids 764-1041 ), VWF(amino acids 764-1045), VWF(amino acids 764-1 128), VWF(amino acids 764-1 198), VWF(amino acids 764-1242), VWF(amino acids 764-1250), VWF(amino acids 764-1261 ), VWF(amino acids 764-1268), VWF(amino acids 764-1464).
  • the amino acid sequence of full length VWF is shown in SEQ ID NO 40.
  • Examples of various fusion partners according to the present invention include:
  • SEQ ID NO 35 - Human serum albumin (the Cys residue in position 34 (shown with bold) may alternatively be substituted with another amino acid such as e.g. Ser or Ala):
  • SEQ ID NO 36 extracellular region of human FcyRI (CD64):
  • SEQ ID NO 38 The C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin (hCG C-terminus):
  • SSSSKAPPPSLPSPSRLPGPSDTPILPQ SEQ ID NO 39: Sequence A / XTEN:
  • VWF Human von Willebrand factor
  • fusion proteins according to the invention include:
  • SEQ ID NO 41 F8-500-A(1649-1657)-LCN-linked-Fc (hlgG1) (B domain deleted/truncated FVIII fused to the Fc domain of human lgG1 in the light chain terminal end at position D1658):
  • SEQ ID NO 42 F8-500-A(1649-1657)-LCN-linked-hCG C-terminus (B domain deleted/truncated FVIII fused to the C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin in the light chain N-terminal end at position D1658):
  • SEQ ID NO 43 F8-500-A(1649-1657)-LCN-linked-albumin (B domain
  • SEQ ID NO 44 F8-500-A(1649-1657)-LCN-linked-FcyRI (B domain
  • SEQ ID NO 45 F8-500-A(1649-1657)-LCN-linked-FVIII(741 -966) (B domain deleted/truncated FVIII fused to a 741 -966 FVIII B domain fusion partner in the light chain N-terminal end at position D1658):
  • compositions and formulations comprising one or more FVIII proteins, preferably fusion proteins of the invention, formulated together with one or more pharmaceutically acceptable carrier(-s).
  • one object of the invention is to provide a pharmaceutical formulation comprising a protein according to the invention present in a concentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0.
  • the formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • a buffer system a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • the pharmaceutical formulation is an aqueous formulation.
  • aqueous formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials.
  • aqueous formulation is defined as a formulation comprising at least 50% w/w water.
  • aqueous solution is defined as a solution comprising at least 50 % w/w water
  • aqueous suspension is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical formulation is a freeze-dried
  • the pharmaceutical formulation comprises an aqueous solution.
  • compositions according to the present invention are preferably suitable for intravenous (IV) and/or extravascular administration (e.g. subcutaneous (sc) or intradermal administration) in prophylactic/therapeutic treatment of haemophilia.
  • IV intravenous
  • extravascular administration e.g. subcutaneous (sc) or intradermal administration
  • Haemophilia Haemophilia/hemophilia/blood clotting diseases is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation ("bleeding disorders"), which is used to stop bleeding when a blood vessel is broken.
  • Haemophilia A clotting factor VIII deficiency
  • Haemophilia A is the most common form of the disorder, present in about 1 in 5,000-10,000 male births. List of embodiments:
  • Embodiment 1 A FVIII fusion protein comprising, or consisting of, a FVIII molecule
  • the FVIII fusion protein according to the invention may furthermore comprise a linker between the FVIII protein and the fusion partner. Amino acids E1649-S1657 are thus absent from said FVIII fusion protein.
  • Embodiment 2 A FVIII protein, wherein amino acids E1649-S1657 are deleted from the FVIII light chain N-terminus.
  • Embodiment 3 A FVIII protein, wherein amino acids E1649-D1658 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position Q1659).
  • Embodiment 4 A FVIII protein, wherein amino acids E1649-Q1659 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position E1660).
  • Embodiment 5 A FVIII protein, wherein amino acids E1649-E1660 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position E1661 ).
  • Embodiment 6 A FVIII protein, wherein amino acids E1649-E1661 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position 11662).
  • Embodiment 7 A FVIII protein, wherein amino acids E1649-11662 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position D1663).
  • Embodiment 8 A FVIII protein, wherein amino acids E1649-D1663 are deleted from the FVIII light chain N-terminus (a FVIII fusion protein, wherein the fusion partner is inserted at the FVIII light chain N-terminus at position Y1664).
  • Embodiment 9 A FVIII fusion protein according to the invention, wherein said fusion protein comprises a fusion partner selected from the group consisting of: albumin (preferably human serum albumin), an Fc domain, an Fc receptor (preferably FcyRI or the extracellular domain/region of FcyRI), and a FVIII B domain fusion partner (preferably amino acids 741 -966).
  • the fusion proteins according to the invention may have (or comprise or consist of) the sequence as set forth in e.g. SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, or SEQ ID NO 44.
  • Embodiment 10 A FVIII protein/fusion protein according to the invention, wherein the B domain of said FVIII molecule comprises 4-908 or 15-25 amino acids.
  • Embodiment 11 A FVIII protein/fusion protein according to the invention, wherein the FVIII B domain fusion partner is a truncated FVIII B domain comprising at least 200 amino acids and up to 500 amino acids.
  • Embodiment 12 A FVIII protein/fusion protein according to the invention, wherein said FVIII molecule comprises, or has, a B domain amino acid sequence/linker of 15-25 amino acids.
  • the sequence of said FVIII B domain is selected from the amino acid sequence in the group consisting of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.
  • Embodiment 13 A FVIII fusion protein according to the invention, wherein said FVIII molecule comprisesan amino acid sequence selected from the list consisting of: SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 1 1 , SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21 , SEQ ID No 22, SEQ ID No 23, SEQ ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31 , SEQ ID No 32, SEQ ID No 33, SEQ ID No 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, S
  • Embodiment 14 A FVIII fusion protein according to the invention, wherein said FVIII B domain fusion partner has a low degree of secondary and tertiary structures.
  • Secondary and tertiary structure can be measured by e.g. Circular Dichroism (CD). Based on e.g. a CD wavelength spectrum from 190-260 nm, wherein the fingerprint of the FVIII B domain fusion partner resembles that of a disordered polypeptide having a signal minimum of 204 nm.
  • the FVIII B domain fusion partner is homogenous in structure in in vivo circulation upon administration to the patient in need thereof.
  • An example of a protein according to the invention having a homogenous structure is a FVIII protein fused to the FVIII B domain fusion partner according to SEQ ID NO 1 1 .
  • Embodiment 15 A pharmaceutical composition comprising a FVIII protein/fusion protein according to the invention.
  • Embodiment 16 A method for making a FVIII protein/fusion protein according to the invention, wherein said method comprises the step of incubating a host cell under appropriate conditions, wherein said host cell comprises an expression vector that encodes a FVIII protein/fusion protein according to the invention.
  • Embodiment 17 A nucleotide molecule encoding a FVIII protein/fusion protein according to the invention.
  • Embodiment 18 An expression vector comprising a nucleotide molecule according to the invention.
  • Embodiment 19 A host cell comprising an expression vector according to the invention.
  • Embodiment 20 A FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, for use as a medicament for treatment of haemophilia.
  • Embodiment 21 A FVIII protein/fusion protein according to the invention, or a pharmaceutical composition according to the invention, for use in treatment of haemophilia by subcutaneous administration.
  • Embodiment 22 A FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, for use in treatment of haemophilia by intravenous administration.
  • Embodiment 23 A method of treatment of haemophilia, wherein said method comprises administering the FVIII protein/fusion protein according to the invention, or a pharmaceutical formulation according to the invention, to a patient in need thereof.
  • a FVIII B domain deleted construct was expressed in CHO cells, wherein the DNA sequence encoding the full length B-domain (aa 741 -1648) has been replaced with a DNA sequence encoding an 21 amino acid residue truncated B-domain with the SEQ ID NO 2.
  • the resulting B domain deleted/truncated FVIII molecule is named turoctocog alfa.
  • An expression vector encoding turoctocog alfa was thus inserted into a CHO host cell for recombinant production thereof.
  • the resulting recombinant turoctocog alfa protein was subsequently purified as previously described (Thim et al, Haemophilia 2010; 16: 349-359) and subjected to N- terminal amino acid sequences analysis.
  • This analyse was carried out by automated Edman degradations using an Applied Biosystem Model 494 Protein Sequencer essentially as described by the manufacturer: the protein is degraded by the removal of one amino acid residue at a time starting from the N-terminal of the protein. The removed amino acid residue is converted to phenylthiohydantoin (PTH) amino acid derivative and subsequently identified and quantified by HPLC analysis.
  • PTH phenylthiohydantoin
  • Table 1 Automated Edman degradation of purified turoctocog alfa. Yields of the individual PTH-amino acids are given in each cycle. Numbers in brackets indicate that the same amino acid occurs from more than one sequence.
  • HC heavy chain.
  • LC light chain.
  • the heavy chain of turoctocog alfa was found to be a uniform product having the expected (and encoded) N-terminal sequence.
  • the light chain was found to consist of two species: (i) one species having the N-terminal sequence of full length light chain and (ii) one species having a sequence starting at amino acid residue number 10 of the light chain.
  • the short form of the light chain was found in approx. 33% of the molecules.
  • the degree to which the shorter form is produced appears to be non- predictable and to depend on e.g. the type of FVIII construct, the nature of the mammalian cell (CHO, BHK, HEK, etc.), cell culturing conditions (e.g. Fill and Draw process versus Alternating Tangential Flow (ATP) process), etc.
  • FVIII fusion proteins were produced using constructs encoding FVIII with the light chain having N-terminus at E1649.
  • FVIII samples were loaded on 7% Tris- Acetate gels (NuPAGE, Novex, Life technologies) with 7 lU/lane for coomassie-stained gels, 0.5 lU/lane for silver staining or 0.1 lU/lane for Western blots.
  • Gel electrophoresis was performed according to the manufacturer's instructions, and either Coomassie stained using SimplyBlue Safestain (Invitrogen, Life Technologies), silver stained using SilverXpress (Invitrogen, Life Technologies) or blotted to nitrocellulose membrane using iBIot (Invitrogen, Life Technologies). The blots were blocked 1 h with 3% skim milk in TBS/Tween (50 mM
  • Tris 150 mM NaCI, pH 7.5, containing 0.1 % Tween 20
  • the blot was washed 3 times with TBS/Tween, incubated with peroxidalse-labelled rabbit anti-mouse IgG (Dako), washed and developed using Super Signal West Pico chemiluminescent substrat (Pierce) as described by the manufacturers.
  • the gels were scanned using HP Scanjet G2710 and bands on gels and the blot quantified using CLIQS 1 D software
  • FVIII fusion protein with the fusion partner attached to the N-terminal of the LC had a band with the same mobility as the LC of FVIII without any fusion partner attached
  • FVIII activity of the rFVIII compounds in cell culture supernatant were measured in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: a FVIII standard e.g. rFVIII (turoctocog alfa, Novoeight ® ) calibrated against the 7th international FVIII standard from NIBSC (2009 # 07/350),were diluted to 5 - 4 - 3 - 2 - 1 - 0.5 - 0.25 mll/mL in Coatest assay buffer (50 mM Tris, 150 mM NaCI, 1 % BSA, pH 7.3, with preservative).
  • Coatest SP reagents Chroctocog alfa, Novoeight ®
  • Cell culture supernatants were diluted minimum 10-fold in the same buffer. At least two dilutions were analysed. Fifty ⁇ of samples, standards, and buffer negative control were added to 96-well microtiter plates in duplicates.
  • the factor IXa/factor X reagent, the phospholipid reagent and CaCI 2 from the Coatest SP kit were mixed 5:1 :3 (vol:vol:vol) and 75 ⁇ of this added to the wells. After 15 min incubation at room temperature 50 ⁇ of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 5 min at room temperature before 25 ⁇ 1 M citric acid, pH 3, was added.
  • the absorbance at 405 nm was measured on a microtiter plate reader with absorbance at 620 nm used as reference wavelength.
  • the value for the negative control was subtracted from all samples and calibrators and the activity of the samples calculated based on a calibration curve prepared by plotting the absorbance values vs. FVIII concentration of the FVIII calibrator dilutions.
  • the FVIII antigen concentration was determined in an ELISA (Factor VIII antigen (FVIILC), Affinity Biologicals) using a polyclonal anti-FVIII antibody both for catching and detection.
  • Microtiter plates (96 well plates, NUNC Maxisorp) were coated overnight at 4°C with 100 ⁇ capture antibody from the kit (F8C-EIA-C). After 5 times washing in PBS + 0.05 % Tween 20 and 15 min incubation with the buffer at room temperature the wells were blocked 30 min at room temperature with 3% casein.
  • FVIII calibrator (turoctocog alfa, Novoeight ® ) diluted to 100 - 33 - 1 1 - 3.70 -1.23 - 0.41 - 0.14 ng/mL (corresponding to 600 - 200 - 67 - 22 - 7.4 - 2.5 - 0.8 pM) in 0.1 M Hepes, 0.1 M NaCI, 10 g/L BSA and 0.1 % Tween 20, pH 7.0, and buffer negative control added in duplicates to the wells and incubated 1-1.5 h at room temperature.
  • the specific activity of the FVIII fusion protein was calculated by dividing the activity of the samples with the FVIII antigen concentration. From table 2 below it can be seen that the activity of the LC-N linked FVIII fusion protein with the fusion partner attached to amino acid 1658 (the deltal 649-1657 light chain) had maintained FVIII activity. The molar specific activity was 67% of FVIII without fusion partner. The specific activity of FVIII in cell culture supernatant is lower than when measured on purified protein (see below) likely due to the presence of excess light chain in the cell culture supernatant.
  • the protein concentration can be determined in an ELISA as described above or by HPLC by applying approximately 10 ⁇ g FVIII fusion protein on a Daiso 300 A, 5 mm, 2.1 x 250 mm column (FeF Chemicals A/S) equilibrated with 1 % trifluoro acetic acid (TFA) at a flow of 0.25 mL/min and a temperature of 40°C using an Agilent 1 100 instrument. The protein was eluted with a 30 min gradient of 35-84% of 80% acetonitrile in 0.09 % TFA.
  • the concentration of the sample was determined by integrating the area under the peaks determined at 214 nm and compare with the area of the peaks in a parallel analysis of a rFVIII protein (turoctocog alfa, Novoeight ® ), where the concentration was determined by amino acid analyses. The molar concentration is calculated by dividing the concentration in mg/mL with the molecular weight of the protein.
  • Table 3 includes the protein concentration of the LC-(delta1649-1657)-N-linked FVIII fusion protein by ELISA. The protein concentration by ELISA was in agreement with the protein concentration determined by HPLC (2.6 mg/mL corresponding to 15705 nM).
  • the specific molar activity of the purified FVIII fusion protein was determined by dividing the activity with the molar protein concentration.
  • the data shown in table 3 are based on concentration determination by ELISA. The data demonstrate that the specific molar activity was maintained for the FVIII fusion protein with fusion partner attached to the LC lacking the first nine amino acids.
  • Example 5 FVIII fused with a fusion partner at the N-terminus of FVIII light chain:
  • Human serum albumin (SEQ ID NO 35) inserted between amino acid 1648 and 1649 of the "F8-500 FVIII molecule with the B domain linker sequence according to SEQ ID NO 2 (SEQ ID NO 46).
  • the extracellular region of human FcyRI (CD64 - SEQ ID NO 36) inserted between amino acid 1648 and 1649 of the "F8-500” FVIII molecule (SEQ ID NO 47).
  • Human FVIII amino acids 741 -966 (SEQ ID NO 1 1 ) inserted between amino acid 1648 and 1649 of the "F8-500” FVIII molecule (SEQ ID NO 48).
  • SEQ ID NO 46 F8-500-LCN-linked-albumin (B domain deleted/truncated FVIII fused to human albumin in the light chain N-terminus end at position D1649):
  • SEQ ID NO 47 F8-500-LCN-linked-FcgRI (B domain deleted/truncated FVIII fused to extracellular region of FcgRI in the light chain N-terminal end at position D1649):
  • SEQ ID NO 48 F8-500-LCN-linked-FVIII(741-966) (B domain
  • SEQ ID NO 49 F8-500-LCN-linked-Fc (hlgGl) (B domain
  • Example 6 FVIII fused with a fusion partner at the N-terminus of FVIII light chain without FVIII amino acid 1649-1657:
  • CHOEBNALT85 cell cultures were transiently transfected with plasmids encoding the fusion proteins of Examples 5+6. Portions of 1 X 107 CHOEBNALT85 cells in 700 ul 1 :1 CD CHO and SFM II medium
  • Thermo Fisher Scientific were electroporated with 10 ug plasmid in a Gene Pulser Cuvette (BioRad) using a GenePulser Xcell (Biorad). Subsequently, the cells were transferred to 125 ml Erlenmeyer flasks with 30 ml of the above growth medium and incubated in a shaker incubator at 36.5 °C and 8 % C02. Five days after transfection, the culture volumes were increased 20 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific), and the incubation temperature was reduced to 30 °C.
  • cell culture supernatants were collected and frozen in aliquots with 20 uM imidazol pH 7.0 (Sigma) and 0.2 % Tween 80 (Merck). The supernatants were utilized for Western blotting as described below.
  • a vial with supernatant from each of the transfected cultures were thawn and 7.5 ul of each supernatant were transferred to individual tubes along with 2.5 ul 4 X NuPage LDS sample buffer (Thermo Fisher Scientific) and 1.1 ul 10 X NuPAGE sample Reducing Agent (Thermo Fisher Scientific).
  • the tubes were incubated for 10 minutes at 70° C on a block heater and then centrifuged 20.000 X g for 2 minutes.
  • the content of each tube was loaded in the wells of a NuPAGE 4-12 % Bis-Tris gel with 15 wells (Thermo Fisher Scientific).
  • the samples were electrophorized in the gel for 2 hours at 200 volt in MOPS running buffer (Thermo Fisher Scientific) with NuPAGE Antioxidant (Thermo Fisher Scientific) added to the running buffer in the inner electrophoresis chamber.
  • the gel was blotted for 7 minutes in an iBIot module (Thermo Fisher Scientific) to a nitrocellulose membrane using the iBIot Gel Transfer Stacks
  • the membrane were washed 4 times for 3 minutes at room temperature with gentle agitation in TBS with 0.1 % Tween 20. Then, the membranes were incubated for 45 minutes as described above but protected against light with donkey anti- sheep Alexa fluor 680 conjugate (Molecular Probes) diluted 1 :10000. Subsequently, the membranes were washed as describesd above 4 times for 3 minutes in TBS with 0.1 % Tween 20. Finally, the membranes were scanned in an Odyssey reader at 680 nM.
  • the FVIII heavy chain of all FVIII proteins migrated as protein with a molecular weight between the 80 kDa and 100 kDa molecular weight markers.
  • the wild-type FVIII light chain of F8-500 was visualized as 4 bands with higher electrophoretic mobility than the FVIII HC.
  • the wild-type FVIII light chain consists of 4 individual bands to due to N-terminus heterogeneity (amino acid E1649 or D1658) and due to incomplete utilization of a glycosylation site at N1810. For each of the fusion proteins containing FVIII amino acid 1649 to1657 (the fusion proteins in Example 5), two FVIII light chain bands were detected.
  • One light chain band migrated between the 100 kDa and 220 kDa molecular weight markers and represented FVIII light chain with fusion partner.
  • the other light chain band co- migrated with wild-type FVIII light chain and represented FVIII light chain without fusion partner.
  • the FVIII light chain without fusion partner constituted 1 1.4 - 23.2 % of the total amount of FVIII light chain.
  • bands of FVIII light chain without fusion partner were not seen for the fusion proteins without FVIII amino acid 1649-1657 (the fusion proteins in Example 6).

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Abstract

La présente invention concerne des molécules de fusion du facteur VIII, ainsi que des compositions pharmaceutiques qui conviennent pour le traitement des maladies de coagulation du sang.
PCT/EP2016/072414 2015-09-22 2016-09-21 Protéines de fusion du facteur viii WO2017050820A1 (fr)

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CN110950964A (zh) * 2018-09-26 2020-04-03 安源医药科技（上海）有限公司 突变型单链人凝血因子viii融合蛋白及其制备方法与用途

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