WO2015021993A1 - Molécules sb101 modifiées pharmacologiquement actives et leurs utilisations - Google Patents

Molécules sb101 modifiées pharmacologiquement actives et leurs utilisations Download PDF

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WO2015021993A1
WO2015021993A1 PCT/EE2014/000005 EE2014000005W WO2015021993A1 WO 2015021993 A1 WO2015021993 A1 WO 2015021993A1 EE 2014000005 W EE2014000005 W EE 2014000005W WO 2015021993 A1 WO2015021993 A1 WO 2015021993A1
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protein
fusion protein
amino acid
acid sequence
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PCT/EE2014/000005
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Anne Pink
Priit Kogerman
Andres Valkna
Aili KALLASTU
Marianna SKOLNAJA
Taavi Pall
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Ibcc Holding As
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70585—CD44
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Sequence listing This application contains sequence data provided on computer readable format and a paper version.
  • the paper version of the sequence data is identical to the data provided on computer readable format.
  • the invention relates to pharmacologically active molecules for use in inhibiting angiogenesis. More specifically the invention relates to pharmacologically active SB 101 molecules and their use. Background of the invention
  • Angiogenesis is a fundamental step in development of larger tumors and in the transition of tumors from benign stage to malignant. For this reason inhibitors of angiogenesis have been considered to have a potential to constitute a treatment for cancer diseases, as well as for a number of other angiogenesis related diseases.
  • CD44 is a cell surface receptor for the large glycosaminoglycan of the extracellular matrix hyaluronic acid (HA) and can also interact with other ligands, such as matrix metalloproteinases (MMPs). CD44 plays a role in various cellular and physiological functions, including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis.
  • HA extracellular matrix hyaluronic acid
  • MMPs matrix metalloproteinases
  • W099/45942 discloses the use of HA-binding proteins and peptides including CD44 to inhibit cancer and angiogenesis related diseases.
  • Ahrens et al. discloses that soluble CD44 inhibits melanoma tumor growth by blocking the cell surface CD44 binding to hyaluronic acid.
  • Bajorath 2000 Provides CD44 and its binding to HA, cell adhesion and CD44-signaling.
  • Bartolazzi et al. 1994 discloses an experiment where mammalian cell expressed CD44HRg-molecule inhibits tumor in nude mice, but a mutant molecule CD44-R41A-Rg, expressed in mammalian cells does not mediate attachment to HA and does not have similar inhibiting effect on the tumor.
  • CD44-3MUT does not have the HA-binding capacity but that it does inhibit angiogenesis and endothelial cell proliferation. It was also shown that CD44-3MUT does not bind to VEGF receptors in vitro, as does VEGF and therefore, the anti-angiogenic effect of CD44-3 MUT is most probably not due to orthosterical or allosterical inhibition of VEGF binding to its receptor.
  • SB 101 is used herein as a synonym for CD44 -3MUT
  • SBS 101 is used herein as a synonym for CD44 -3MUT
  • the molecule has vimentin dependent activity in suppressing cell- cell adhesion.
  • Another object of the invention is to provide modified SB 101 -molecules having extended in vivo plasma half-life times.
  • VEGFR2 by administering SBIOI-Fc fusion protein having an amino acid sequence of SEQ ID NO: 4.
  • a further object of the invention is to provide a method to inhibit angiogenesis in vivo by administering bacterial cell expressed pegylated SB 101 having an amino acid sequence of SEQ ID NO:2 or of SEQ ID NO:6.
  • a yet another object of the invention is to provide a method to inhibit tumor growth in a mammal in vivo by administering SBIOI-Fc fusion protein.
  • Still another object of the invention is to provide a method to block endothelial cell response to VEGF- stimulation by administering bacterial cell expressed pegylated SB 101 having an amino acid sequence of SEQ ID NO:2 or of SEQ ID NO:6.
  • Yet another object of the invention is to provide an expression vector comprising a nucleotide sequence substantially according of SEQ ID NO: 3 encoding SBIOI-Fc fusion protein, together with control elements enabling the expression of said nucleotide sequences in a host cell.
  • a further object of the invention is to provide a method to inhibit growth of a tumor by introducing amino acid sequence according to SEQ ED NO: 2, SEQ ID NO:4 or SEQ ID NO:6 into tumor cells.
  • CD44-3MUT To increase serum residence time of CD44-3MUT two different approaches were used - in one case bacterially expressed CD44-3MUT was modified by PEGylation and alternatively, CD44-3MUT was expressed and purified in mammalian system as human IgGl Fc region fusion protein. Both approaches surprisingly resulted in biologically active protein with significantly increased in vivo serum half-life. Altogether, CD44-3MUT direct PEGylation or its expression as IgGl Fc fusion protein significantly improves its pharmacokinetic properties.
  • Figure 1 illustrates expression of SB 101 and purification of inclusion bodies (IB).
  • A Sequence alignment of amino acid residues 27-132 of human CD44 (SEQ ID NO:5) and SB 101 (SEQ ID NO:6) proteins. The positions of mutated residues in SB 101 (R41A, R78S and Y79S) are indicated by upward arrows. Secondary structure elements are shown on top of sequence alignment and are based on crystal structure of CD44 HABD, 1UUH (Teriete, P.S. et al., 2004, Mol Cell 13: 483-96). Right arrows, beta-structures. Helices, alpha-helices and 3 10 -helix.
  • SB 101 (B, left panel) SDS-PAGE analysis of SB 101 expression.
  • SB 101 was transformed into E.coli BL21-CodonPlus(OE3)-RP strain and expressed in MAI for 22 h at 37°C.
  • SB 101 (indicated by arrow) expression was estimated by analyzing samples from pre-culture and final culture (total lysate) on SDS-PAGE.
  • SB 101 expressing cells were lysed and soluble fraction was separated from inclusion bodies (IB) by centrifugation. After washing (IB washes), IBs were solubilized in 8 M urea containing buffer and centrifuged to separate insoluble IB fraction. SB 101 containing soluble IB lysate was further used for SB 101 purification.
  • Figure 2 illustrates purification of SB 101.
  • IB Ion exchange chromatogram of urea dissolved inclusion bodies
  • Soluble IB lysate was loaded onto HiPrepTM 16/10 DEAE FF 20 ml column and bound proteins were eluted by stepwise and linear gradient of NaCl.
  • Eluates from FT and from first and second peak (PI and P2, respectively) were collected throughout IEC and analyzed on SDS-PAGE (B).
  • Eluates containing monomeric SB 101 shown by arrow
  • IEC pool GF chromatogram of IEC purified SB101.
  • IEC pool containing SB101 (load) was applied onto HiLoadTM 26/60 SuperdexTM 200 preparation grade column.
  • Figure 3 illustrates PEGylation of SB 101.
  • A SDS-PAGE analysis of SB 101 PEGylation reaction. SB 101 was conjugated with 20 kD methoxy-PEG-propionaldehyde. PEG, SB 101 and PEGylation reaction mixture (mixture) were analysed by SDS-PAGE.
  • B SDS-PAGE analysis of purified PEG-SB 101. PEG-SB 101 was purified from reaction mixture by IEC (Mono Q 4.6/100 PE column). SDS-PAGE gels were co-stained with Coomassie brilliant blue R-250 and PEG specific Bal 2 (A and B left panels) or Coomassie brilliant blue alone (A and B right panels).
  • FIG. 4 illustrates plasma half-life of SB 101. Rats were injected intravenously with PEG-SBlOl, SBlOl or GST-SB 101. Protein plasma levels were measured by ELISA assay (see Materials and methods), which was carried out in triplicates. Data are represented as mean ⁇ SE. As non-PEGylated proteins display very short plasma half-life, these are also plotted on the graph with shorter time scale (insert).
  • Figure 5 illustrates comparison of SB lOl and PEG-SB 101 functional activity by endocytosis and cell layer impedance assay.
  • CHOEBNALT85 supernatant (pQMCF-5) 48 h after transfection.
  • B) illustrates coomassie blue stained SDS-PAGE analysis of purified SBIOI-Fc.
  • Figure 7 illustrates plasma half-life of SBIOI-Fc. Rats were injected intravenously with SB IOI-Fc. Protein plasma levels were measured by ELISA assay. Data represent mean of triplicate wells ⁇ 95% CI.
  • Figure 8 illustrates inhibition of in vivo angiogenesis in mice by SBIOI-Fc intraperitoneal administration at doses 0.5 to 25 mg/kg.
  • A) illustrates experimental scheme, where basement membrane extract filled angioreactors containing premixed FGF2, VEGF and heparin or PBS alone for uninduced controls were implanted SC into flanks of nude mice.
  • mice started to receive every second day via IP injections of SB IOI-Fc, control human IgGl-Fc or vehicle (PBS). After 14 days, angioreactors were resected and the population of endothelial cells within the angioreactor matrix was assessed by FITC-lectin staining. The number of fluorescent cells was quantitated by micro plate reader. Background subtracted raw readings from each experiment were scaled by division with their root mean square.
  • B) illustrates summary of directed in vivo angiogenesis assay results, bargraphs show mean ⁇ SD. N, number of independent experiments.
  • P values were calculated from post-hoc pairwise comparisons using t tests with pooled SD.
  • P value adjustment method fdr. Effect sizes were calculated by Cohen's d formula (95% confidence intervals). Confidence intervals were calculated using bootstrapping.
  • GF growth factors (FGF2/VEGF).
  • B) illustrates SBIOI-Fc blocking endothelial cell response of VEGF stimulation.
  • N 3 independent experiments. HUVEC were seeded onto 96WE1 ECIS arrays (Applied Biophysics, Troy, NY, USA). Next day, cells were changed into 0.5% FBS containing starvation media. After overnight serum starvation, cells were incubated 1 h with indicated amounts of SBIOI-Fc or rhlgGl-Fc and thereafter released from serum starvation by addition of 25 ng/ml VEGF in presence of recombinant protein. Cell growth was continuously monitored by measuring impedance at seven different frequencies from 1000 Hz to 64000 Hz using ECIS ⁇ instrument (Applied BioPhysics).
  • FIG. 10 Shown is impedance data collected at Zlk, Z4k, Z16k and Z64k, impedance at 1000 Hz, 4000 Hz, 16000 Hz and 64000 Hz, respectively. Error bars, standard deviation.
  • Figure 10 illustrates SBIOI-Fc causing inhibition of VEGFR2 Tyrosine-1175 phosphorylation.
  • A) illustrates western blotting with anti-pVEGFR2(Tyrll75) and anti- VEGFR2 antibodies.
  • B) illustrates densitometric quantitation of pVEGFR2(Tyrll75) and total VEGFR2 western blots from three or five experiments, respectively.
  • HUVEC were seeded to 6-well culture plate at density 80000 cells/well.
  • FIG. 11 Angiogenic response in mouse strains from different genetic backgrounds. Angiogenesis assay and quantitation was performed essentially as described in Example 7. Error bars, standard deviation.
  • CD44-3MUT also called SB-101
  • CD44-3MUT does not have the HA-binding capacity but that it does inhibit angiogenesis and endothelial cell proliferation.
  • CD44-3MUT does not bind to VEGF receptors in vitro, as does VEGF and therefore, the anti-angiogenic effect of CD44-3MUT is most probably not due to orthosteric or allosteric interference with VEGF binding to its receptor.
  • SBlOl-protein (synonymous to CD443MUT) contains amino acid residues 27-132 of human CD44 and carry amino acid substitutions R41A, R78S and Y79S.
  • the sequence of SB 101 (SEQ ID NO: 6) is shown in Figure 1A in comparison with the sequence of residues 27-132 of CD44 (SEQ ID NO: 5).
  • a longer variation of the protein (SEQ ID NO: 2) where the N-terminus has an additional MQIDLNI-sequence was also used.
  • Both of truncated (SEQ ID NO: 6) and the non-truncated version (SEQ ID NO:2) are referred here as SB 101 -protein.
  • the initial concept was to provide pharmacologically active modified SB 101 molecules.
  • Recombinant bacterially expressed SB 101 exhibits very short plasma half-life in vivo.
  • the goal for this study was to improve the pharmacokinetic properties of recombinant SB101.
  • the goal was to introduce mammalian cell expressed SB 101 -protein derivatives with an ability to inhibit angiogenesis.
  • Such molecules would be novel and non-obvious, because all previously published information teaches that glycosylated non HA-binding CD44 mutants would not have the effect of inhibiting angiogenesis.
  • Pegylation is a process of attaching the strands of the polymer PEG to peptides or proteins. Protein pegylation increases the molecular weight of the molecule and can give significant pharmacological advantages over unmodified forms, such as increased drug , stability and improved drug solubility. Additionally, pegylation masks protein surface, and reduces protease degradation and immunogenicity.
  • SB 101 was cloned and expressed in E.coli cells.
  • the recombinant SB 101 isolated was necessarily in non-glycosylated form.
  • the recombinant SB 101 did not have the HA binding capability, but it still is capable of inhibiting angiogenesis.
  • Bacterial cell expressed SB 101 (SEQ ID NO: 2 or SEQ ID NO:6) was pegylated and unexpectedly it showed similar biological activity as the non-glycosylated non modified form. As is shown below in Example 4 the pegylated SB 101 was capable of inhibit endothelial cells similarly to unmodified SB 101. Analysis of the bacterial cell expressed pegylated SB 101 molecule indicated not only that the pegylated protein was biologically active, but it also showed that the pharmacokinetics of this molecule were superior to the unmodified SB 101: The half life time of the pegylated SB 101 is over 70 times longer than the half life time of the unmodified molecule. Comparing the molecular weights of the unmodified molecule and the pegylated molecule it was recognized that increase in molecular weight of -4.6 times lead to increase in the half life time over 70 times.
  • GST-SB 101 fusion protein did not show longer half life time than unmodified SB 101 protein.
  • SBIOI-Fc showed about 11 times higher half life time than the unmodified SB 101 several times smaller half life time than the pegylated bacterial expressed SB101.
  • pegylated bacterial cell express SB 101 is provided with a half-life time approximately 70 time higher than of unmodified SB 101.
  • a SB 101 -fusion protein is provided with at least 10 times higher half life time than of unmodified SB 101.
  • modified pegylated bacterial cell expressed SB 101 protein is used to treat tumors, cancers and other angiogenesis dependent diseases.
  • mammalian cell expressed fusion protein SBIOI-Fc is used to treat tumors, cancers and other angiogenesis dependent diseases.
  • the modified SB 101 proteins can be used in the treatment of solid tumors, brain cancer, breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, bladder cancer, malignant lymphoma, and pancreatic cancer.
  • the modified SB 101 proteins are administered intraperitoneally to a mammal at doses between 1 and 50 mg kg, preferably between 5 and 25 mg kg.
  • the modified SB 101 proteins are administered several times per week.
  • a pharmaceutical formulation comprising a pharmaceutically acceptable compound of the present invention, which provides, upon administration to a subject (e.g., a human), a decrease in tumor burden.
  • the formulation may be administered in any suitable means including gene therapy.
  • Yet another aspect of the invention is a method of treating ovarian cancer in a subject (e.g., a human) in need thereof by administering to the subject an effective amount of the compound or the pharmaceutical formulation of the present invention.
  • Yet another aspect of the invention is a method of treating colon cancer in a subject (e.g., a human) in need thereof by administering to the subject an effective amount of the compound or the pharmaceutical formulation of the present invention.
  • Yet another aspect of the invention is a method of treating breast cancer in a subject (e.g., a human) in need thereof by administering to the subject an effective amount of the pharmaceutical formulation of the present invention.
  • Yet another aspect of the invention is a method of treating colon cancer before or after surgical resection and/or radiation therapy, in a subject (e.g., a human) in need thereof by administering to the subject an effective amount of the compound or the pharmaceutical formulation of the present invention.
  • Yet another aspect of the invention is a method of treating cancer before or after surgical resection and or radiation therapy, in a subject (e.g., a human) in need thereof by administering to the subject an effective amount of the compound or the pharmaceutical formulation of the present invention, including adjunctive therapy with one or more additional therapeutic agents, or their pharmaceutically acceptable salts.
  • the compounds of the invention may also be administered sequentially with known anticancer or cytotoxic agents when a combination formulation is inappropriate. In any combination treatment, the invention is not limited in the sequence of administration.
  • the amount and frequency of administration of the compounds of the invention will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated.
  • SB 101 was cloned into Ndel/Hindlll site of pETllc vector (Novagen, Darmstadt, Germany). SB 101 was codon optimized by ProteinExpert (SEQ ID NO: 1. Grenoble, France). For protein expression, SB 101 -vector was transformed into E.coli BL21-CodonPlus(DE3)- RP strain. For preculture, 3-4 colonies of transformants were inoculated into 20 ml of autoinduction media (MAI; Studier FW, 2005, Protein Expr Purif 41(1): 207-34) containing 100 ⁇ g ml ampicillin and 34 ⁇ g/ml chloramphenicol. The preculture was grown at 37° C overnight (ON) at 200 rpm until OD600 reached -10.
  • SB 101 was mostly expressed in insoluble protein fraction, as there was no SB 101 present in soluble fraction ( Figure IB). Therefore, we purified SB 101 from inclusion bodies (IB).
  • the ON thawed cell pellet was resuspended in Buffer A, 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 2 mM MgCl 2 , 150 mM NaCl, and 1 mM PMSF, by using a Dounce hand homogenizer.
  • the buffer volume was 5 ml/g cells.
  • Cell suspension was supplemented with 100 ⁇ g/ml lysozyme (Sigma) and 10 ⁇ g/ml DNase I (Sigma) and incubated with continuous stirring for 2 h at 4° C. Cells were lysed using French press 3 times at 18000 psi.
  • IB Inclusion bodies
  • Wash buffer I 50 mM Tris-HCl pH 8.0, 2 M NaCl, 0.1% Triton X-100, 1 mM PMSF.
  • Wash buffer II 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 1 M urea, 1 mM PMSF.
  • Wash buffer III, IV and V 50 mM Tris- HC1 pH 8.0, 150 mM NaCl, 1 M urea, 1 mM PMSF.
  • the volume of IB wash and dissolving buffer was calculated as 12 ml per gram of starting cell mass.
  • IBs were dissolved in Buffer B, 50 mM Tris-HCl, pH 8.0, 8 M urea, 25 mM ethylenediamine (EN), 1 mM PMSF, ON with continuous strirring at 4° C and insoluble debris was pelleted by centrifugation at 46000xg for 30 min at 4° C. After dissolving isolated IBs in 8 M urea-buffer and removal of insoluble material, soluble IB lysate contained considerable amount of SB 101 ( Figure IB).
  • IEC Ion exchange chromatography
  • SB 101 protein was eluted in FT and peak 1 and peak 2 contained only little amount of SB 101. While SB 101 in FT appeared to be in its monomeric state, peak 1 and 2 contained mostly aggregated or oligomeric SB101 as assessed by SDS-PAGE analysis under nonreducing conditions (Figure 2B).
  • EEC pool For further purification we pooled SB 101 containing FT and termed it as EEC pool.
  • Peak 3 contained considerable amount of pure monomeric SB 101 as found by SDS-PAGE under reducing and non-reducing conditions ( Figure 2C and 2D).
  • Fractions containing purified monomeric SB 101 and used this pool in refolding steps were pooled.
  • monomeric protein containing fractions were pooled and diluted up to 0.1 mg/ml with GF buffer.
  • Refolding was performed by stepwise dialysis from 8 M urea solution into PBS. Each dialysis step was performed twice, except last PBS step which was performed six times, for 1 h on a magnetic stirrer at 4°C.
  • the sequentially used dialysis solutions were I: 6 M urea, 50 mM Tris-HCl pH 8.0, 18mM EN; II: 4 M urea, 50 mM Tris-HCl pH 8.0, 12 mM EN; III: 2 M urea, 50mM Tris-HCl pH 8.0, 150mM NaCl, 6mM EN; IV: 1 M urea, 50mM Tris-HCl pH 8.0, 150mM NaCl, 3mM EN; V: 0.5 M urea; 50mM Tris-HCl pH 8.0, 150mM NaCl, 1.5mM EN; VI: 50mM Tris-HCl pH 8.0, 150mM NaCl; VII PBS pH 7.4.
  • MALDI-TOF MS peptide fingerprinting showed that both of these bands contained SB 101. Therefore, it was assumed that these bands represent different conformations of SB 101. However, it was non possible to separate these putative differently folded forms neither by IEC nor GF.
  • Calculated average mass of SB 101 is 11497.8 Da, assuming that the N-terminal methionine of the protein is excised.
  • MALDI-TOF MS analysis of SB101 showed that the average molecular mass of the protein was 11500.4 Da (mass difference +2.6 Da; relative error 226 ppm; Figure 2G).
  • MALDI-TOF MS fingerprinting of Glu-C or trypsin digested SB 101 was performed. Fingerprinting results showed that Glu-C and trypsinolysis peptide spectra covered 100% of SB 101 sequence (Table 1). Glu-C digestion of SB 101 also confirmed the excision of N- terminal methionine.
  • Purified SB 101 of Example I was conjugated with 20 kD methoxy-PEG-propionaldehyde (Jenkem Technology USA, Allen, TX, USA) in the presence of 200-fold molar excess of NaB3 ⁇ 4CN as reducing agent.
  • the reaction was performed in PBS pH 7.4 for 24 h. After 6 h and 22 h the reaction mixture was supplemented with additional dose of reducer in 100-fold and 50-fold molar excess, respectively.
  • reaction mixture was dialyzed against 10 mM phosphate buffer by three consecutive steps and then loaded onto a strong anion exchange column Mono Q 4.6/100 PE (GE Healthcare) equilibrated with lOmM phosphate buffer pH 7.4. After washing the column with 5 column volumes (CV) of lOmM phosphate buffer, PEGylated SB 101 was eluted by linear gradient of 0-1 M NaCl in 10 mM phosphate buffer. Reaction mixture was analysed by SDS-PAGE stained with Coomassie brilliant blue, or PEG-specific Bal 2 (Figure 3A). Expected molecular weight of monoPEG-SB lOl was 31.6 kD.
  • test proteins were quantified by sandwich ELISA using anti-SB 101 mAb 1A2.H4 for GST-SB 101 and SB 101 capture and anti-PEG mAb for PEG-SB 101 capture.
  • Anti-SB 101 pAb and anti- SB lOlmAb were used as detection antibodies, respectively.
  • Vectastain ABC kit Vector Laboratories, Burlingame, CA, USA
  • tetramethylbenzidine substrate were used for colour development and absorbance was measured using ELISA plate reader (Tecan, Mannedorf, Swizerland). Plasma concentrations of tested proteins were interpolated from the standard curves generated by using serums with known concentrations of proteins. The obtained protein plasma concentration time curves are shown in Figure 4 (3D).
  • Plasma concentration time-curves were fitted to exponential one-phase decay model using GraphPad Prism 5 software (version 5.02; Dec- 17, 2008; GraphPad Software, Inc.) and calculated pharmacokinetic properties are summarized in Table 2.
  • Pharmacokinetic analysis showed that unmodified SB 101 proteins, regardless of their size and purification method, were rapidly cleared from rat circulation.
  • PEGylation of SB 101 significantly prolonged its half-life in rat circulation.
  • PEG coupling to SB 101 extended its plasma half-life about 70- fold.
  • PEGylation also considerably increased the systemic exposure (as measured by AUC) and respectively decreased the plasma clearance of SB 101 when compared to unmodified proteins. All tested proteins displayed similar volume of distribution values from 2.4-4.4% of total body weight, which indicates that all these proteins confined to plasma water.
  • SB101 To assess PEGylated SB101 in vitro activity, we investigated its cellular uptake similarly to the method described in (Pall T., A. Pink, L. Kasak, et al., 2011; PLoS ONE 6: e29305).
  • MLEC mouse lung endothelial cells
  • MLECs were seeded into 0.1% gelatin coated 8- well microscopy slides at density 20000 cells/well. MLECs were incubated 10 min with 0.11 ⁇ /L SB 101 or PEG-SB 101, followed by 20 min chase after changing to fresh media.
  • Cell-bound SB101 was detected using 1A2.H4 mAb and anti- mouse-Alexa 488 conjugated secondary Ab (Molecular Probes).
  • DNA sequence encoding SB 101 was amended with sequence according to SEQ ID NO: 20 encoding N-terminal Cystatine-S signal peptide sequence (SEQ ID NO:21 ) and synthesized by Genewiz, Inc.
  • the obtained synthetic DNA was inserted N-terminally into pBluescript vector containing human IgGl Fc region sequence (SEQ ID NO: 22) encoding IgGlFc region according to SEQ ID NO:23.
  • the resulting SBIOI-Fc cDNA (SEQ ID NO 3) was cloned into pQMCF-5 expression vector containing RSV-LTR promoter (vector from Icosagen Cell Factory, Estonia).
  • the resulting expression plasmid was transfected into CHOEBNALT85 cells.
  • Protein expression was analyzed 48 h after transfection from cell media by Western Blot analysis (Figure 6A).
  • Anti-human IgG-HRP conjugate (1:5000; LabAs, Estonia) was used for detection of protein expression.
  • Example 3 above for SB 101 and SB101-PEG except that 250-300 g male F344/NCrHsd rats (Harlan, Netherlands) were used and 3 mg of SBIOI-Fc was administered via tail vein injection or into jugular vein catheter. Plasma concentration of SBIOI-Fc was measured using sandwich ELISA. 96- well plate (Maxisorp, Nunc) was coated with mouse anti-human IgGl antibody clone G17-1 (BD Biosciences) 4 ⁇ g ml in 100 ⁇ coating buffer (CB; 15mM Na 2 C0 3 , 35mM NaHC0 3) pH 9) per well. Plates were blocked with 1.5% BSA in PBS 1 h at RT.
  • CB coating buffer
  • SBIOI-Fc standards were serially diluted (40 ⁇ - 0 ⁇ g/ml) into buffer containing 0.5% BSA and 2% rat serum in PBS. Serum samples collected from different time points were diluted 1:50 in 0.5% BSA in PBS. Biotin-conjugated mouse anti human IgG antibody clone G18-145 (BD Biosciences) 2 ⁇ in 1% BSA in PBS was used for detection. After detection antibody incubation wells were washed 6 times with 200 ⁇ 0.05% Tween-PBS and incubated with streptavidin-HRP (BD Biosciences) diluted 1: 1000 in 0.5% BSA in PBS 30 min at RT in dark.
  • Plasma concentrations of SB IOI-Fc were interpolated from the standard curve generated by using rat serums with known concentrations of protein.
  • the protein plasma concentration time curve obtained is shown in Figure 7.
  • Plasma concentration time-curves were fitted to exponential one-phase decay model using GraphPad Prism 5 software (version 5.02; Dec- 17, 2008; GraphPad Software, Inc.) and calculated pharmacokinetic properties are summarized in Table 2.
  • PEG-SB 101 133.7 812.5 81.3 1.2 4.3 171.2 (111 to 372) 0.8701
  • Co is drug plasma concentration at time zero after iv administration.
  • AUC is area under curve, expressed also as percent of initial dose (%ID).
  • CL is clearance.
  • Vd is volume of distribution, expressed as percent of total body weight (%TBW).
  • T 2 is half-life.
  • CI is confidence interval.
  • R2 is coefficient of determination.
  • SBIOI-Fc exhibited intermediate plasma half- life value compared to SB101 and PEG-SB 101. This is surprising finding, given that SB101 Fc- fusion protein apparent molecular weight in its native dimeric form is ⁇ 130 kD ( Figure 6), meaning that it's ⁇ 2.6 times higher than PEG-SB 101 ( Figure 3B and C). Nevertheless, SBIOI-Fc plasma half-life showed at least 10-fold increase over SB 101 half-life. Surprisingly, SBIOI-Fc displayed approximately two times higher volume of distribution (Vd) compared to bacterially produced SB101 molecules.
  • Vd volume of distribution
  • SBIOI-Fc Vd 9.6% (95% CI 9.1 - 10.2) of total body weight suggests that this protein binds also to erythrocytes in addition to plasma water residence.
  • SBIOI-Fc showed increased systemic exposure (AUC) and respectively decreased plasma clearance compared to unmodified SB 101 (Table 2).
  • IV- administration route data show that SBIOI-Fc displays clearly prolonged plasma half-life, but surprisingly less than expected based on its molecular size. SBIOI-Fc displays also an increased apparent volume of distribution.
  • SBIOI-Fc inhibits in vivo angiogenesis In order to test in vivo anti- angiogenic effect of SB 101 and SBIOI-Fc, Directed in
  • Vivo Angiogenesis Assay kit (DIVAATM; Trevigen, USA) was used according to manufacturer's instructions.
  • angiogenesis is measured by blood vessel invasion into tumor extracellular matrix filled angioreactors. Blood vessel in growth is stimulated by combination of two tumor-related angiogenic factors bFGF and VEGF premixed into the angioreactor's matrix.
  • 20 ⁇ angioreactors (semi closed silicone cylinders) were filled with growth factor reduced basement membrane extract (BME) containing premixed bFGF, VEGF and heparin for the induction of angiogenic response.
  • BME growth factor reduced basement membrane extract
  • BME growth factor reduced basement membrane extract
  • bFGF and VEGF in angioreactor's matrix were 1.4 ⁇ / ⁇ 1 and 0.47 ⁇ 1, respectively.
  • Angioreactors were implanted sc into dorsolateral flank of nine week old Hsd:Athymic Nude-Foxnl/nu female or male mice (Harlan, Netherlands) through 0.5-1 cm skin incisions. Implantation was performed on both flanks and two angioreactors were inserted per flank. Incisions were closed with absorbable 6-0 sutures (Monosyn; B Braun, Germany) and 7 mm skin clip (Autoclip; BD Diagnostics, USA). The surgical procedure was carried out under general anesthesia.
  • mice were given ip anesthetic mix of ketamine 75 mg/kg (Ketamin; Farmak, Ukraine) with medetomidine 1 mg/kg (Dorbene Vet; Pfizer/SYVA, Spain). Anesthesia was then reversed by 1 mg/kg atipamezole subcutaneous injection (Antisedan; Pfizer/Orion Pharma, Finland). All steps of angioreactor filling and implantation were performed under aseptic conditions and in accordance with Trevigen DIVAATM protocol. Mice were also given 5 mg/kg of ketoprofen sc injection for postoperative pain relief (Anafen; Merial, France).
  • mice were treated with SB IOI-Fc or vehicle (PBS) during two weeks starting from the next day post implantation ( Figure 8A).
  • the protein according to SEQ ID NO:5 to exemplify the effect, but minor modifications on the protein sequence are in the scope of this invention.
  • the SB IOI-Fc protein has a sequence at least 80% similar to SEQ ED NO:5 and more preferably about 95% similarity with SEQ ID NO:5.
  • Drugs were administered by intraperitoneal injections every second day.
  • mice were sacrificed by carbon dioxide asphyxiation and angioreactors were dissected. Angioreactor contents were retrieved and processed for quantization of endothelial cell invasion according to manufacturer's protocol.
  • BME was digested with metalloproteinase-based CellSperseTM reagent, endothelial cell receptors were then recovered by incubating cells in 10% FBS containing DMEM, then washed several times with wash buffer, repeatedly resuspending cells. Recovered cells were pelleted by centrifugation at 250 x g for 5 min at RT.
  • FITC- Lectin staining was used for fluorometric quantitation of angioreactor-invaded endothelial cells. Cell-bound fluorescence was read at 485 nm excitation and 535 nm emission wavelengths using Tecan microtiter plate reader.
  • SB IOI-Fc treatments don't differentiate significantly - 25 mg/kg dosing shows medium sized effect compared to 0.5 or 5 mg/kg, but lower boundary of 95% CI-s of effect size is in both cases close to zero ( Figure 8C).
  • SB lOl-Fc-mediated angiogenesis inhibition could be achieved by continous delivery via micro osmotic pumps.
  • Alzet 100 ⁇ osmotic pumps, filled in sterile conditions with SBIOI-Fc (0.3 mg/lOO ⁇ ) or rhlgG-Fc (0.3 mg/lOO ⁇ ) were implanted through a midline abdominal incision into intraperitoneal cavity of mouse, anesthetized with a combination of ketamine (Bioketan, 75 mg/kg) and medetomide (Dorbene Vet, 1 mg/kg). Wound was closed in two layers using 6-0 monofilament absorbable sutures (Monosyn;
  • mice received also subcutaneously analgesic ketoprofen (Ketofen, 5 mg/kg). Results show that angiogenesis was inhibited in mice carrying SBlOl-Fc-filled pumps compared to rhlgG-Fc control treatment ( Figure 8B).
  • HUVEC were seeded at density 5000 cells/well. After 30 hours cells were switched to starvation media— M199 supplemented with 1% FBS, 25 mM Hepes and 4 mM L-glutamine for 16 hours. After starving, cells were treated with different concentrations of hlgG-Fc or SBIOI-Fc in 155 ⁇ of 5% FBS containing HUVEC growth media (5% FBS, M199, 4 mM L-glutamine, 12.5 ⁇ heparin, 10 mM Hepes, 7.5 ⁇ g/ml ECGS) for 1 hour at 37°C. Thereafter the cells were stimulated with 25 ng/ml of VEGF.
  • SB 101 binds to cell surface vimentin and that SB 101 is endocytosed and it inhibits cells to VEGF stimulation it is suggested that SB 101 may affect cell growth factor receptor level or activation.
  • SB 101-Fc-treatment assessed the effect of SB 101-Fc-treatment on VEGF receptor 2 protein level and activation.
  • HUVEC cells (passage 4-6, Cell Applications, Inc.) were cultured in 6-well cell culture plates in 20% FBS containing M199 medium supplemented with 4 mM L-glutamine, 50 ⁇ g/ml heparin, 10 mM Hepes, 30 ⁇ g/ml ECGS (Millipore). Cells were seeded at density 80000 cells/well. After 24 hours cells were starved in M199 media supplemented with 1% FBS, 25 mM Hepes and 4 mM L-glutamine for overnight.
  • the membranes were then incubated with primary antibodies in 2% whey- TBST (5% BSA-TBST for phospho-antibodies) overnight at 4°C followed by appopriate HRP- conjugated secondary Ab (Jackson Immunoresearch) incubation at 1/10000 dilution for 1 hour at RT. After every Ab incubation step the membranes were washed 4 x 10 min with TBST at RT. For reprobing, the membranes were incubated in stripping buffer (62.5 mM Tris pH 6.8, 2% SDS, 100 mM 2-mercaptoefhanol) for 30 min at 50°C. Results show that 1 hour treatment of endothelial cells prior VEGF stimulation with SB IOI-Fc causes dose dependently inhibition of VEGFR2 activation at phospho-Tyrosine 1175 ( Figure 10).
  • DIVAATM Directed in Vivo Angiogenesis Assay kit
  • angiogenesis is measured by blood vessel invasion into tumor extracellular matrix filled angioreactors. Blood vessel in growth is stimulated by combination of two tumor-related angiogenic factors bFGF and VEGF premixed into the angioreactor's matrix.
  • 20 ⁇ angioreactors are filled with growth factor reduced basement membrane extract (BME) containing premixed bFGF, VEGF and heparin for the induction of angiogenic response.
  • BME growth factor reduced basement membrane extract
  • BME containing equal volume of PBS was used for filling angioreactors.
  • Final concentrations of bFGF and VEGF in angioreactor's matrix are 1.4ng ⁇ l and 0.47ng ⁇ l, respectively.
  • Angioreactors are implanted sc into dorsolateral flank of nine week old Hsd:Athymic Nude- Foxnl/nu female or male mice (Harlan, Netherlands). Implantation is performed on both flanks and two angioreactors are inserted per flank. Mice are treated with PEG-SB 101 at doses 5 and 25 mg/kg or vehicle (PBS) during two weeks starting from the next day post implantation. Drugs are administered by intraperitoneal injections every second day.
  • mice are sacrificed and angioreactors are dissected. Angioreactor contents are retrieved and processed for quantization of endothelial cell invasion according to manufacturer's protocol.
  • FITC -Lectin staining is used for fluorometric quantization of angioreactor-invaded endothelial cells.
  • Cell-bound fluorescence is read at 485 nm excitation and 535 nm emission wavelengths using Tecan microtiter plate reader.
  • PEG-SB 101 effect on angiogenesis by intraperitoneal administration at tested doses will be compared to vehicle treated controls and mean treatment effects are subjected to statistical analysis.
  • Example 11 Testing PEG-SB101 effect on endothelial cell response to VEGF stimulation
  • Real-time monitoring of endothelial growth in impedance based assay is used to test whether PEG-SB 101 can interfere with mitogenic stimulation of endothelial cells.
  • Growing cells cover the surface of gold electrodes of the measurement arrays resulting in increased electrical impedance (i.e. complex resistance).
  • HUVEC are seeded onto 8WE1 ECIS arrays (Applied BioPhysics, Troy, NY, USA).
  • Next day cells are changed into 0.5% FBS containing starvation media. After overnight serum starvation, cells are supplemented with 10% FBS and PEG-SB 101 or vehicle (PBS) alone. After 1 hour PEG-SB 101 and vehicle (PBS) treated cells are further stimulated by supplementing media with 5 ng/ml VEGF.
  • VEGF mitogenic stimulation causes increase in cells impedance.
  • PEG-SB 101 treatment is expected to block such VEGF effect similarly to SB101 or SB IOI-Fc.
  • Real-time monitoring of endothelial growth in impedance based assay is used to test whether SB 101, SBIOI-Fc or PEG-SB101 can interfere with stimulation of endothelial cells with growth factors.
  • SB 101, SBIOI-Fc or PEG-SB 101 interferes with hepatocyte growth factor (HGF/SF) stimulation of endothelial cells.
  • HGF/SF hepatocyte growth factor
  • HGF/SF stimulates endothelial cell growth, motility and angiogenesis by binding to the c-Met receptor and may confer resistance to anti VEGF/VEGFR pathway cancer therapies (Shunli Ding, Tatyana Merkulova-Rainon, Zhong Chao Han and Gerard Tobelem, BLOOD, 2003 101: 4816-4822; Shojaei F, Lee JH, Simmons BH, Wong A, Esparza CO, Plumlee PA, Feng J, Stewart AE, Hu-Lowe DD, Christensen JG, CANCER RES., 2010, 70: 10090-100).
  • impedance assay growing cells cover the surface of gold electrodes of the measurement arrays resulting in increased electrical impedance (i.e.
  • HUVEC are seeded onto 8WE1 ECIS arrays (Applied BioPhysics, Troy, NY, USA). Next day, cells are changed into 0.5% FBS containing starvation media. After overnight serum starvation, cells are supplemented with 10% FBS and SB lOl, PEG-SBlOl, SBIOI-Fc or vehicle (PBS) alone. After 1 hour SB lOl, PEG-SB lOl, SBIOI-Fc and vehicle (PBS) treated cells are further stimulated by supplementing media with 25 ng/ml HGF. Cell growth is continuously monitored over next 24-48 hours by measuring impedance at 100- 64000 Hz using ECIS ⁇ instrument (Applied BioPhysics). HGF stimulation causes changes in cells impedance. SB lOl, PEG-SBlOl, SB IOI-Fc treatments is expected to block such HGF effects compared to control (vehicle or irrelevant protein) treatments.
  • Real-time monitoring of endothelial growth in impedance based assay is used to test whether SBlOl, SB IOI-Fc or PEG-SBlOl can interfere with growth factor stimulation of endothelial cells.
  • Growth factors stimulate endothelial cell growth, motility and angiogenesis by binding to their receptors.
  • growth factors involved in angiogenesis include TGF- alpha and FGF-2 and other factors which bind to their receptors
  • impedance assay growing cells cover the surface of gold electrodes of the measurement arrays resulting in increased electrical impedance (i.e. complex resistance).
  • HUVEC are seeded onto 8WE1 ECIS arrays (Applied BioPhysics, Troy, NY, USA).
  • cells are changed into 0.5% FBS containing starvation media. After overnight serum starvation, cells are supplemented with 10% FBS and SBlOl, PEG-SB lOl, SB IOI-Fc or vehicle (PBS) alone. After 1 hour SB lOl, PEG-SB lOl, SBIOI-Fc and vehicle (PBS) treated cells are further stimulated by supplementing media with selected growth factor. Cell growth is continuously monitored over next 24-48 hours by measuring impedance at 100-64000 Hz using ECIS ⁇ instrument (Applied BioPhysics). Growth factor stimulation causes changes in cells impedance. SBlOl, PEG-SB lOl, SB IOI-Fc treatments is expected to block such growth factor effects compared to control (vehicle or irrelevant protein) treatments.
  • the endothelial cells are treated 1 hour with SB IOI-Fc, SBlOl or PEG-SB 101 prior growth factor stimulation.
  • TGF-alpha and FGF-2 are used for stimulation.
  • Growth factor stimulation causes rapid endocytosis of growth factor receptors. Endocytosed receptor is either recycled back to the plasma membrane or targeted to lysosomes for degradation. After stimulation, respective growth factor receptor protein levels are measured using western blotting.
  • SBlOl, PEG-SBlOl, SB IOI-Fc treatments is expected to reduce growth factor receptor levels in endothelial cells in this assay.
  • Example 13 CD44 deficiency associates with increased angiogenic response
  • FGF2/VEGF- induced angiogenic response in CD44 knockouts and wild type mice from C57BL/6, C3H or mixed genetic backgrounds ( Figure 11).
  • p 0.0052
  • N 2 experiments
  • effect size 13.5 95% CI 13.5 to Inf
  • p 0.41

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Abstract

La présente invention concerne des molécules SB101 modifiées ayant des caractéristiques pharmacocinétiques améliorées. La protéine pégylée a une demi-vie plus longue que la protéine de fusion SB101-Fc recombinante exprimée dans des cellules de mammifère. SB101 pégylé présente une demi-vie plasmatique in vivo 70 fois plus longue et la protéine de fusion présente une demi-vie approximativement 10 fois plus longue que SB101 non modifié. La protéine pégylée et la protéine de fusion ont maintenu leur activité in vitro similaire à SB101 non modifié. Les molécules de protéine de fusion de SB101 inhibent l'angiogenèse, bloquent la réponse des cellules endothéliales à VEGF et inhibent la protéine de récepteur de VEGF 2. Les molécules modifiées ayant des caractères pharmacocinétiques améliorés sont des candidats pour le traitement de maladies associées à l'angiogenèse telle que le cancer.
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WO2019166644A1 (fr) * 2018-03-02 2019-09-06 Mimetas B.V. Dispositif permettant d'effectuer des mesures électriques
CN112203765A (zh) * 2018-03-02 2021-01-08 米梅塔斯私人有限公司 用于进行电测量的设备
CN112203765B (zh) * 2018-03-02 2022-12-13 米梅塔斯私人有限公司 用于进行电测量的设备

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