Inhibition of platelet aggregation using anti-human GPVI antibodies

Abstract

The present invention relates to an isolated humanized protein binding to human Glycoprotein VI (hGPVI) for treating a GPVI-related condition in a subject in need thereof, wherein said isolated humanized protein is to be administered during at least 2 hours to the subject, preferably during at least 4 to 6 hours.

Claims

1. A method for treating a cardiovascular disease or event associated with inflammation and/or thrombosis in a subject in need thereof comprising administering an isolated humanized protein binding to human Glycoprotein VI (hGPVI), wherein said isolated humanized protein is administered for at least 2 hours, wherein said isolated humanized protein is a monovalent antibody fragment, wherein the variable region of the heavy chain of the antibody fragment comprises the following CDRs: TABLE-US-00018 VH-CDR1: (SEQ ID NO: 1) GYTFTSYNMH; VH-CDR2: (SEQ ID NO: 2) GIYPGNGDTSYNQKFQG; and VH-CDR3: (SEQ ID NO: 3) GTVVGDWYFDV; and the variable region of the light chain of the antibody fragment comprises the following CDRs: TABLE-US-00019 VL-CDR1: (SEQ ID NO: 4) RSSQSLENSNGNTYLN; VL-CDR2: (SEQ ID NO: 5) RVSNRFS; and VL-CDR3: (SEQ ID NO: 6) LQLTHVPWT.

2. The method according to claim 1, wherein said isolated humanized protein is administered during at least 4 to 6 hours.

3. The method according to claim 1, wherein the isolated humanized protein is injected.

4. The method according to claim 1, wherein the isolated humanized protein is administered by intravenous infusion.

5. The method according to claim 1, wherein a dose of humanized protein ranging from about 0.5 mg/kg to about 50 mg/kg is administered to the patient.

6. The method according to claim 1, wherein a dose of humanized protein ranging from about 1 mg/kg to about 32 mg/kg is administered to the patient.

7. The method according to claim 1, wherein a dose of humanized protein of about 8 mg/kg is administered to the patient.

8. The method according to claim 1, wherein a dose of humanized protein ranging from about 125 mg to about 2000 mg is administered to the patient.

9. The method according to claim 1, wherein a first bolus is administered.

10. The method according to claim 9, wherein the first bolus comprises about 10 to 50% of the total dosage of the isolated humanized protein to be administered.

11. The method according to claim 9, wherein said first bolus is administered in about 5 to 30 minutes.

12. The method according to claim 1, wherein said protein binds to a conformational epitope comprising: at least one amino acid residue from amino acid residues 114 to 142 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 114 to 142 of hGPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 165 to 187 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 165 to 187 of hGPVI (SEQ ID NO: 13).

13. The method according to claim 12, wherein said conformational epitope comprises at least one amino acid residue from amino acid residues 121 to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 121 to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169 to 183 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 169 to 183 of hGPVI (SEQ ID NO: 13).

14. The method according to claim 1, wherein said protein has a KD for binding to hGPVI less than 15 nM, wherein said K.sub.D is measured by surface plasmon resonance using 960 to 1071 RU of soluble human GPVI and using PBS pH 7.4 as running buffer and wherein said isolated humanized protein does not induce a GPVI depletion phenotype in vivo.

15. The method according to claim 1, wherein said protein is a monovalent antibody fragment selected from the group consisting of a single chain antibody, a Fv, a Fab; and a unibody.

16. The method according to claim 1, wherein the amino acid sequence encoding the heavy chain variable region of the antibody fragment is SEQ ID NO: 7 and the amino acid sequence encoding the light variable region of the antibody fragment is SEQ ID NO: 8, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 8.

17. The method according to claim 15, wherein the amino acid sequence encoding the heavy chain variable region of the antibody is SEQ ID NO: 7 and the amino acid sequence encoding the light variable region of the antibody is SEQ ID NO: 9, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 9.

18. The method according to claim 1, wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from arterial and venous thrombosis, restenosis, acute coronary syndrome, cerebrovascular accidents due to atherosclerosis, critical limb ischemia, cerebral vascular diseases, ischemic stroke, venous thromboembolism diseases, thrombotic microangiopathies and vascular purpura.

19. The method according to claim 1, wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from coronary artery and cerebral artery diseases.

20. The method according to claim 1, wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from atherothrombosis, ischemic events, acute coronary artery syndrome, myocardial infarction, stroke, percutaneous coronary intervention, stenting thrombosis, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches, peripheral artery disease, venous thrombosis, acute phlebitis, pulmonary embolism, cancer-associated thrombosis, inflammatory thrombosis and thrombosis associated to infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1B are a combination of graphs showing the mean±SEM intensity (FIG. 1A) and velocity (FIG. 1B) of collagen-induced platelet aggregation measured 30 minutes and 2 hours after the end of a 15 minutes' infusion of increasing doses of ACT017 (1, 2, 4, 8 mg/kg) to cynomolgus monkeys (n=4).

(2) FIG. 2 is a graph showing the blood platelet count of cynomolgus measured before any treatment (TO) or 24 hours after the infusion of vehicle or ACT017 at increasing doses (1, 2, 4, 8 mg/kg).

(3) FIG. 3 is a combination of graphs showing the bleeding time measured in 4 subjects, before treatment (Time=0) and 30 min and 2 hours after the infusion of ACT017 (1, 2, 4, 8 mg/kg) or vehicle to cynomolgus monkeys.

(4) FIGS. 4A-4B are a combination of graphs showing the mean±SD intensity (FIG. 4A) and velocity (FIG. 4B) of collagen-induced platelet aggregation measured at different time after the beginning of a one-hour infusion of two doses of ACT017 (2 and 8 mg/kg) to cynomolgus monkeys (n=8).

(5) FIG. 5 is a graph showing the level of GPVI expression measured by flow cytometry on the platelets of cynomolgus monkeys (n=8) at different times after the beginning of a one-hour infusion of ACT017 at 2 mg/kg (MFI: Mean Fluorescence Intensity).

(6) FIG. 6 is a graph showing the mean±SD intensity of collagen-induced platelet aggregation measured at different times (from 20 minutes to 24 hours), after the end of a 25-minute bolus injection of ACT017 8 mg/kg (n=4), a 1-hour infusion of ACT017 8 mg/kg (n=8), or a 15-minute bolus of ACT017 2 mg/kg followed by a 5-hour-45-minute infusion of ACT017 6 mg/kg to cynomolgus monkeys (n=4).

(7) FIG. 7 is a graph showing the binding of wild-type GPVI-Fc (black graph) and GPVI double mutant (grey graph) on collagen.

(8) FIG. 8 is a graph showing the binding of ACT017 on wild-type GPVI-Fc (black graph) or on double mutant (grey graph).

(9) FIG. 9 is a graph showing the percentage of platelet aggregation measured with two healthy subjects of cohort 4 at different times (from 15 min to 168 hours). Subjects of cohort 4 were administered with 500 mg of ACT017 or of placebo. The “screening time” represents the measurement of the percentage of platelet aggregation one week or 24 hours before the administration of ACT017 or its matching placebo. The “pre-dose” represents the baseline measurement of the administration of the ACT017 or its matching placebo. In black is presented the graph for healthy subject 1 and in grey is presented the graph for healthy subject 2.

EXAMPLES

(10) The present invention is further illustrated by the following examples.

Example 1: Biologic Data in Non-Human Primates

(11) Materials and Methods

(12) Animals

(13) 28 cynomolgus monkeys free of any previous treatment were used in the study.

(14) Treatment

(15) First, increasing doses of ACT017 (1, 2, 4, 8 mg/kg) or its vehicle were intravenously administrated over 15 minutes (n=4-8). At time 30 minutes and 2 hours after administration, blood was collected.

(16) In a second experiment, ACT017 was administered to animals, by bolus injection or infusion. Three treatment groups were included in the study: ACT017 administrated at 8 mg/kg by a bolus injection of 25 minutes (n=4), ACT017 administrated at 8 mg/kg by an infusion of 1 hour (n=8), ACT017 administrated at 2 mg/kg by a 15-min bolus injection followed by a 6 mg/kg infusion of 5 hours and 45 minutes (n=4). At different times, from 20 minutes to 24 hours after administration, blood was collected.

(17) Analysis

(18) Platelet aggregation: Platelet rich plasma (PRP) was obtained from monkeys after centrifugation (120×g; 15 min; 20° C.) and immediately used. The velocity and intensity of the aggregation were measured using the APACT® aggregometer and a collagen concentration of 2.5 mg. mL-1 (Horm collagen, Nycomed, DE) to the PRP containing various concentration of the Fab of the invention and continuously recorded. The intensity of platelet aggregation was measured as the percent increase in light transmission. The mean±SD are presented at the indicated time after the beginning of the injection.

(19) Platelet count was measured in EDTA anticoagulated blood. The platelet count was determined in a Scil Vet abc automatic cell counter (Scil Animal Care Company, Holtzheim, France) set to monkey parameters.

(20) The bleeding time was measured on vigil monkeys at the surface of the forearm, according to standard clinical procedure (Ivy's procedure) and using 0.5 cm Surgicutt™ bleeding time device.

(21) GPVI expression: Blood samples were collected onto EDTA before injection and 30 minutes post injection of increasing doses of ACT017 or an equivalent volume of vehicle and were incubated with commercial FITC coupled anti-human GPVI monoclonal antibody (clone 1G5, Biocytex) that cross react with cynomolgus GPVI, and fluorescence was measured on a Beckman Coulter Gallios Flow cytometer.

(22) Results

(23) The effect of ACT017 administration was characterized in non-human primates. Eight cynomolgus monkeys were enrolled in the study.

(24) First, increasing doses of ACT017 (1, 2, 4, 8 mg/kg) were intravenously administrated over 15 minutes. At time 30 minutes and 2 hours after administration, blood was collected. Platelet aggregation was reversibly inhibited in a dose dependent manner (FIGS. 1A-1B). Increasing the dose from 1 to 2 and from 2 to 4 mg/kg increased the effect whereas 8 mg/kg had no additional effect as compared to 4 mg/kg. The platelet count measured 24 hours after the injection was not modified as compared to the values obtained before injection (FIG. 2). No significant increase in the bleeding time was observed after treatment with vehicle or 2, 4 or 8 mg/kg ACT017 (FIG. 3). Next, after a washing out period, the cynomolgus received ACT017 (2 or 8 mg/kg) administered as a one-hour perfusion. Platelet aggregation and GPVI expression were measured at different time after the beginning of the treatment: (1, 1.5, 2, 4 and 7 hours) (FIGS. 4 and 5). Collagen-induced platelet aggregation was reversibly inhibited and the duration of the effect was prolonged in cynomolgus treated with 8 mg/kg compared to 2 mg/kg (FIGS. 4A-4B). GPVI expression on platelets remained stable at the different time after the beginning of the treatment as compared to the pretreatment values (FIG. 5).

(25) Together, these results confirm in non-human primates that administration of ACT017 efficiently and reversibly inhibits GPVI function without impact on the platelet count, on expression of GPVI at the platelet surface nor on bleeding time.

(26) In addition, according to the preceding results, the inhibitory effect of ACT017 on platelet aggregation, after a 1-hour infusion appeared to be efficient during a limited period of time. Indeed, ACT017 induced a pronounced and stable inhibition on platelet aggregation only during 2 hours after the beginning of the administration, i.e. for at most 1 hour after the end of the 1-hour induction (see FIGS. 4A-4B). After 2 hours, even if the inhibition was prolonged at 8 mg/kg, ACT017 inhibitor effect regressed at all tested doses.

(27) In a second experiment the effect of ACT017 at the dose of 8 mg/kg was further characterized on platelet aggregation after different administration times, including 1-hour and 6-hour administrations. Platelet aggregation was reversibly inhibited in a time dependent manner (FIG. 6).

(28) A 25-minute bolus injection at 8 mg/kg induced a full inhibition of the collagen-induced platelet aggregation measured at 0.5 hour. At 2 hours, the inhibition of platelet aggregation regressed and the aggregation intensity returned to values of 24±34%. No effect of ACT017 can be observed at 24h.

(29) A 1-hour infusion at 8 mg/kg resulted in a prolonged inhibition of collagen-induced platelet aggregation. However, the inhibition was total (<5%) for all animals only until 2 hours. At 4 hours and 7 hours after ACT017 administration, inhibition regressed and the overall mean intensity of aggregation was respectively 16±29% and 25±32%. Therefore, as for the 25-min administration, the inhibitory effect of ACT017 appears to be pronounced only during a short time after its 1-hour administration.

(30) A 6-hour infusion at 8 mg/kg resulted in a profound inhibition of collagen-induced platelet aggregation lasting at least 9 hours. Indeed, during 9 hours after the beginning of the 6-hour infusion, platelet aggregation intensity was lower than 2%. Moreover, the possibility of a longer duration of the effect is not excluded since no analysis was performed between 9 hours and 24 hours. At 24 hours, the effect of ACT017 was close to be fully reversed for three out of the four animals and the mean intensity of aggregation reached 47±30%.

(31) In conclusion, for the same dose of 8 mg/kg, a 6-hour infusion demonstrates a prolonged efficacy to inhibit collagen-induced platelet aggregation during at least 3 hours after the end of the administration.

Example 2: Study of ACTO17 Safety, Tolerability, Pharmacokinetic and Pharmacodynamic in Healthy Volunteers

(32) Materials and Methods

(33) The present study is a randomized, double blind, placebo-controlled ascending single dose study on the safety, tolerability, pharmacokinetics and pharmacodynamics of ACT017 in healthy volunteers.

(34) Subjects

(35) The subjects included in the study were healthy male or non-pregnant, non-breastfeeding female subject, aged between 30 and 60 year of age (inclusive) with a BMI≥18 kg/m2 and ≤30 kg/m2. A total of 48 subjects were enrolled in 6 ascending dose level cohorts with each cohort consisting of 8 subjects: 6 on active and 2 on placebo. Each cohort was divided into 2 sub-cohorts: one cohort dosed initially (1 active and 1 placebo) and the other cohort (5 active and 1 placebo) 48 hours thereafter.

(36) On Day −1 of each dosing period, subjects were admitted to the research center and stay there until at least 48 hours' post-dose. A follow up visit was paid on Day 7. Subject were hospitalized from Day-1 until the morning of Day-3.

(37) Treatment

(38) Investigational drugs were 50 mL vial containing 500 mg of ACT017 and matching placebo solution. Treatments were given during an infusion of 6 hours.

(39) Each dose was given as a 6-hour infusion, with a 15 minutes injection of a first bolus corresponding to about ¼ of the final dosage.

(40) Cohort 1: Treatment A: 62.5 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(41) Cohort 2: Treatment A: 125 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(42) Cohort 3: Treatment A: 250 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(43) Cohort 4: Treatment A: 500 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(44) Cohort 5: Treatment A: 1000 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(45) Cohort 6: Treatment A: 2000 mg ACT017 (n=6), Treatment B: matching placebo (n=2).

(46) Analysis

(47) Pharmacokinetic blood sampling for ACT017 implied 15 samples per subject.

(48) Urine collection for ACT017 implied approximately 5 collection intervals per subject.

(49) Safety and tolerability analysis included the following studies: Adverse events: throughout the study; Vital signs: frequently; ECG (electrocardiogram): less frequent than vital signs; Clinical laboratory including hematology: 2 times; Coagulation parameters: 5 times; Bleeding time: 4 times; Platelet count: 7 times; Platelet aggregation (collagen): 6 times; Immunogenicity/ADA: 3 times.

(50) Platelet aggregation: Platelet rich plasma (PRP) was obtained from healthy subjects having received the placebo or 500 mg of ACT017 intravenously with 25% of the dose administered in the first 15 min an 75% of the dose administered in the following 5 h 45 min, after centrifugation (120×g; 15 min; 20° C.) and immediately used. The intensity of the aggregation were measured using the APACT® aggregometer and a collagen concentration of 2.5 mg. mL-1 (Horm collagen, various concentration of the Fab of the invention and continuously recorded. The intensity of platelet aggregation was measured as the percent increase in light transmission.

(51) Results

(52) As shown by FIG. 9, the percentage of aggregation of platelets in subject 2 decreases by about 70% only 15 min after the beginning of the administrating in comparison with the baseline percentage and is maintained at about 10% for the next 8 hours, then it is increased again to reach about 70% of aggregation after 24 hours, and then returns to baseline 48 hours after administration. No effect is seen on platelet aggregation 168 hours after administration. The effect observed in subject 2 is thus reversible. In subject 1, the percentage of platelet aggregation seems unchanged during the 168 hours of the experience.

Example 3: Study of Epitopes Affinity of ACT017 on GPVI

(53) The epitope of ACT017 on GPVI was previously identified by epitope mapping as being a conformational epitope comprising two regions on GPVI: amino acids 121-135 and amino acids 169-183 in SEQ ID NO: 13. In order to validate this epitope, a double mutant was constructed, comprising the following mutations: S125P, S126Q, G128R, Q133K, T136S, T171D, A172L and H174V and the affinity of this mutant for collagen and ACT017 was measured.

(54) Materials and Methods

(55) Soluble GPVI-Fc was produced as follows: a gene encoding the ectodomain of human GPVI from the first methionine to asparagine 269 fused to the human IgG1 Fc domain via the tripeptide GGR was synthesized after codon optimization. This gene was cloned into the pTT5 vector before being transfected into HEK 293-6E cells. Secreted GPVI-Fc was purified from the conditioned media of the cells by affinity chromatography using MAbselect matrix (GE Healthcare, 17519901) followed by a polishing chromatography on Nuvia™ HR-S cation exchange resin (BioRad, 156051).

(56) Effect of Double Mutant on Binding of GPVI-Fc on Collagen—

(57) Microtitration plates were coated with Collagen in PBS (20 μg/mL, 100 μL per well) overnight at 4° C.

(58) Nonspecific binding sites were saturated with 200 μL of 1% BSA in PBS for 30 min. The plates were then incubated with increasing concentrations of wild-type or double mutant GPVI-Fc preparations (100 μL in PBS containing 0.1% BSA and 0.1% Tween 20) for 30 min. After 3 washing rounds plates were incubated with a peroxidase-coupled secondary human anti-Fc(ab) for 30 min. Finally, 100 μL of the substrate solution were added to each well for 4 min, and the colorimetric reaction stopped by 25 μL NaOH 3 M.

(59) Effect of Double Mutant on Binding of ACT017 on GPVI-Fc—

(60) Microtitration plates were coated with GPVI-Fc in PBS (20 μg/mL, 100 μL per well) overnight at 4° C. Nonspecific binding sites were saturated with 200 μL of 1% BSA in PBS for 30 min. The plates were then incubated with increasing concentrations of ACT017 preparations (100 μL in PBS containing 0.1% BSA and 0.1% Tween 20) for 30 min. After 3 washing rounds plates were incubated with a peroxidase-coupled secondary human anti-IgG for 30 min. Finally, 100 μL of the substrate solution were added to each well for 4 min, and the colorimetric reaction stopped by 25 μL NaOH 3 M.

(61) Analysis

(62) Absorbance at 450 nm is measured with Flustar Optima.

(63) Results

(64) As shown by FIG. 7, double mutant of GPVI-Fc is still capable to bind to collagen in a dose-dependent manner showing that the affinity of GPVI for collagen is mainly conserved in the presence of these mutations. However, as shown by FIG. 8, ACT017 is capable to bind wild-type GPVI-Fc, but not the double mutant, even at high concentrations. These results thus confirmed the position of the epitope of ACT017 on GPVI sequence.