ANTI-PROTEIN S SINGLE-DOMAIN ANTIBODIES AND POLYPEPTIDES COMPRISING THEREOF
20230265211 · 2023-08-24
Inventors
- François SALLER (Le Kremlin Bicetre Cedex, FR)
- Cécile DENIS (Le Kremlin Bicetre, FR)
- Delphine BORGEL (Le Kremlin Bicetre Cedex, FR)
- Frédéric BORGEL (Le Kremlin Bicetre Cedex, FR)
- Oliver CHRISTOPHE (Le Kremlin-Bicetre, FR)
- Peter LENTING (Le Kremlin-Bicetre, FR)
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
C07K16/38
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
A61P7/02
HUMAN NECESSITIES
International classification
Abstract
Vitamin K-dependent Protein S (PS) is a natural anticoagulant acting as a non-enzymatic cofactor for both activated protein C (APC) and tissue factor pathway inhibitor (TFPI). The inventors identify an anti-PS nanobody that very surprisingly enhances the APC-cofactor activity of PS through unknown mechanisms. Very interestingly, this nanobody exerts an antithrombotic effect in injured mesenteric microvessels of mice. As a consequence, it10 constitutes a novel class of antithrombotic agents that could be used for the treatment of acute microthrombosis in pathological states such as sepsis, COVID-19, distal microvascular thrombosis induced by stroke, or sickle-cell disease. Thus, the present invention relates to isolated single-domain antibodies (sdAb) directed against protein S (PS) and polypeptides comprising thereof.
Claims
1. An single-domain antibody (sdAb) directed against protein S (PS), wherein the single-domain antibody enhances APC-cofactor activity of PS.
2. The single-domain antibody according to claim 1, wherein said single-domain antibody comprises the CDR1 having the sequence set forth as SEQ ID NO: 1, the CDR2 having the sequence set forth as SEQ ID NO: 2 and the CDR3 having the sequence set forth as SEQ ID NO: 3.
3. The single-domain antibody according to claim 1 having at least 70% identity with the sequence set forth as SEQ ID NO 4.
4. The single-domain antibody according to claim 1 which comprises the sequence set forth as SEQ ID NO: 4.
5. A cross-competing single-domain antibody which cross-competes for binding PS with the single domain antibody according to claim 1.
6. A polypeptide comprising at least one single-domain antibody according to claim 1.
7. The polypeptide according to claim 6 which comprises at least two single-domain antibodies which enhance the APC-cofactor activity of PS.
8. The polypeptide according to claim 6, comprising two single-domain antibodies which enhance the APC-cofactor activity of PS.
9. The polypeptide according to claim 7 which comprises the sequence set forth as SEQ ID NO:5.
10. A nucleic acid molecule encoding i) the single-domain antibody of claim 1 and/or ii) a polypeptide comprising at least one single-domain antibody of claim 1.
11. A vector that comprises the nucleic acid of claim 10.
12. A host cell which has been transfected, infected or transformed by j the nucleic acid of claim 10 and/or ii) a vector comprising the nucleic acid of claim 10.
13. A method for preventing or treating a thrombotic disorders in a subject in need thereof, comprising administering to said subject an effective amount of the single-domain antibody of claim 1 and/or a polypeptide comprising at least one single-domain antibody according to claim 1.
14. A method for preventing or treating vaso-occlusive crisis in a subject in need thereof, comprising administering to said subject an effective amount of the single-domain antibody of claim 1 and/or a polypeptide comprising at least one single-domain antibody according to claim 1.
15. The method of claim 13, wherein the thrombotic disorders is sepsis, sickle-cell anemia, embolism, stroke or cardiovascular disease.
16. A pharmaceutical composition comprising the single-domain antibody of claim 1 and/or a polypeptide comprising at least one single-domain antibody according to claim 1.
17. The method of claim 15, wherein the embolism is a pulmonary or cerebral embolism.
18. The single-domain antibody of claim 1, wherein the single-domain antibody is an isolated single-domain antibody.
Description
FIGURES
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[0148]
[0149] The results indicated that PS003biv efficiently bound to rSBHG, and therefore that the epitope of PS003biv is localized within the SHBG-like region of PS. As this region is only found in Gas6, the absence of binding of PS003biv to rhGas6 strongly suggested that PS003biv was specific for PS.
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[0151] The results demonstrated that both PS003 and PS003biv enhanced the APC-cofactor activity of rhPS in our plasma-based assay, and that the enhancing effect of PS003biv on the APC-cofactor activity of rhPS appeared higher than that of PS003.
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EXAMPLE 1
[0157] Material & Methods
[0158] Selection of the PS003 Nanobody by Phage-Display
[0159] Anti-PS nanobodies were identified essentially as previously described for anti-VWF nanobodies (Aymé et al. 2017). Briefly, immunization of a single llama (L. glama) with recombinant human PS (rhPS) was outsourced to the Centre de Recherche en Cancérologie (Université Aix-Marseille, Marseille, France). Blood was collected for the isolation of peripheral blood lymphocyte, and lymphocyte total mRNA was used for the construction of a single-domain antibody (sdAb)-library. Briefly, total mRNA was used for the synthesis of cDNA via reverse transcriptase using a CH2′ primer. sdAb-coding DNA fragments were obtained from the cDNA by a nested-PCR reaction, and fragments were subsequently cloned into the pHEN6 phagemid vector. The ligated material was used to transform competent TG1 E. coli cells (ThermoFischer Scientific) allowing the creation of a library of >107 transformants. Phages exposing each sdAbs were rescued by infection of a culture of this library with M13K07-helper phages, and phage particles were incubated with Dynabeads M-450 epoxy beads coated with purified rhPS (1 mg/mL) for 1 h at room temperature in 50 mM Tris, 150 mM NaCl pH 7.4 (TBS Buffer) containing 2% BSA and 5 mM CaCl2. Magnetic beads were washed nine times with using TBS containing 0.1% Tween-20 and 5 mM CaCl2), and twice with TBS containing 5 mM CaCl2. Captured phages were eluted by incubation with 500 μL of 1 mg/mL trypsin in TBS, for 30 min at room temperature. Eluted phages (500 μL) were diluted in 500 μL of TBS, and 5 μL of eluted phages were serially diluted to infect TG1 E. coli cells, and plated to evaluate the PS-specific enrichment. The remainder of the eluted phage solution was amplified after rescue using M13K07-helper phages to perform a new round of enrichment. Two consecutive rounds of enrichment were performed. After the second round of enrichment, 5 μL of eluted phages were serially diluted to infect TG1 E. coli cells, and plated to obtain single ampicillin-resistant colonies. To isolate genuine PS-specific nanobodies, these TG1-clones were grown overnight in 96-deep-well culture plates in 0.5 mL 2YT-medium and nanobody expression was induced with 1 mM IPTG. Periplasmic extracts containing the nanobodies were prepared as described and tested for binding to immobilized rhPS or BSA in a direct ELISA. This allowed the identification of a strong and specific PS binder, named PS003.
[0160] Construction of PS003 and PS003biv Nanobodies
[0161] To allow intracytoplasmic bacterial expression, the cDNA sequence of PS003 was cloned into the pET28-plasmid between the 5′ PstI and 3′ BstEII restriction sites. In this pET28 format, the protein sequence of PS003 is flanked by an N-terminal His6-tag and a C-terminal haemagglutinin (HA)-tag to facilitate purification and detection (
[0162] Expression and Purification of Nanobodies The plasmids encoding monovalent and bivalent nanobodies were used to transform competent T7 SHuffle E. coli cells (New England Biolabs). For each nanobody, a single kanamycin-resistant colony was cultured at 30° C. in LB medium containing 30 μg/mL kanamycin until 0.4<OD.sub.600 nm<0.6. The cytoplasmic expression of nanobodies was then induced by the addition of 0.1 mM IPTG, and nanobodies were produced for 16 h at 20° C. Bacterial pellets were resuspended in 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4 containing 10 μg/mL lysozyme (Sigma) and 25 U/mL benzonase (Sigma), and supplemented with SigmaFAST protease inhibitors (Sigma). The suspension was sonicated and centrifuged at 12000 rpm for 30 min at 4° C. Lysates were frozen at −20° C.
[0163] Monovalent nanobodies were purified through immobilized metal ion chromatography (IMAC). Briefly, lysates were thawed at 37° C. and centrifuged at 4700 rpm for 30 min at 20° C. Supernatants were loaded at 1 mL/min on a HiTrap TALON Crude column (GE Healthcare) pre-equilibrated in 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4. The column was washed with >20 column volumes of 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4, and washed with >20 column volumes of 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4 containing 10 mM imidazole. The bound nanobodies were eluted with 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4 containing 150 mM imidazole, and fractions (1 mL) were collected. The protein content in each fraction was determined by measuring the OD.sub.280 nm, and the fractions of interest were pooled and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS buffer). The dialysate was eventually concentrated on an Amicon® Ultra-15 centrifugal filter device (3 kDa cut-off) (Merck Millipore).
[0164] Bivalent nanobodies were purified through Protein A affinity chromatography. Briefly, lysates were thawed at 37° C. and centrifuged at 4700 rpm for 30 min at 20° C. Supernatants were loaded at 0.5-1 mL/min on a HiTrap Protein A Fast Flow column (GE Healthcare) pre-equilibrated in 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4. The column was washed with >20 column volumes with 50 mM NaH.sub.2PO.sub.4, 0.3 M NaCl pH 7.4, and the bound nanobody was eluted with 0.1 M Glycine pH 2.7. Fractions (1 mL) were collected in tubes containing 100 μL of 1M Tris.Math.HCl pH 8.5. The protein content in each fraction was determined by measuring the OD.sub.280 nm, and the fractions of interest were pooled and dialyzed against TBS buffer. The dialysate was eventually concentrated on an Amicon® Ultra-15 centrifugal filter device (3 kDa cut-off) (Merck Millipore).
[0165] Epitope Mapping of PS003
[0166] A recombinant form of the PS SHBG-like domain alone (rSHBG) was previously expressed and purified (Saposnik et al. 2003). BSA, purified rhPS and purified rSHBG (60 μL at 8 μg/mL in TBS containing 5 mM CaCl.sub.2)) were immobilized onto 96-well NUNC Maxisorp plates for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 5 mM CaCl.sub.2 and 0.1% Tween-20) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2 and 5% BSA. Wells were washed with 3×200 μL of washing buffer and a fixed concentration of PS003 (2 nM in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at 37° C. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-HA tag antibody (Abcam, 1 μg/mL in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. The results demonstrated that the epitope for PS003 was localized within the SHBG-like domain of PS (
[0167] Results
[0168] Specificity of PS003 for rhPS
[0169] To gain insights into the specificity of PS003, we tested the ability of purified PS003 to bind to PS and to various vitamin K-dependent proteins that contain homologous domains (Gla and EGF-like domains). Recombinant human PS (rhPS), recombinant human FIX (BeneFIX, Pfizer), recombinant human FX (Haematologic technologies), plasma-derived protein Z (Hyphen BioMed), and recombinant human Gas6 (rhGas6) (Clauser et al. 2012) (60 μL at 10 μg/mL in TBS containing 5 mM CaCl.sub.2) were immobilized onto 96-well NUNC Maxisorp plates for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 0.1% Tween-20 and 5 mM CaCl.sub.2) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2) and 5% BSA. Wells were washed with 3×200 μL of washing buffer and a fixed concentration (20 nM in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) of purified PS003 was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-HA tag antibody (Abcam, 1 g/mL in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. The results suggested that PS003 strongly bound only to rhPS (
[0170] Vitamin K-dependent Gas6 is highly homologous to PS (47% overall homology) and, in contrast to other vitamin K-dependent proteins, also contains a SHBG-like domain. To further confirm the specificity of PS003 to PS, rhPS and rh Gas6 (60 μL, at 10 μg/mL in TBS containing 5 mM CaCl.sub.2) were immobilized onto 96-well NUNC Maxisorp plates for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 5 mM CaCl.sub.2 and 0.1% Tween-20) and wells were blocked for 1 h at room temperature in TBS containing 5 mM CaCl.sub.2 and 5% BSA. Wells were washed with 3×200 μL of washing buffer and increasing concentrations of PS003 (0-200 nM in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-HA tag antibody (Abcam, 1 μg/mL in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. The results demonstrated that PS003 strongly and dose-dependently bound to rhPS but not to rhGas6 (
[0171] Binding of Recombinant and Plasma-Derived PS to Immobilized PS003 in Sandwich ELISA
[0172] In our phage-display strategy, PS003 was selected on an immobilized form of rhPS covalently coupled to magnetic beads. In addition, PS003 was found to strongly bind to rhPS immobilized onto ELISA wells. To rule out that PS003 only recognizes a non-native immobilized form of rhPS, the binding of rhPS in solution to immobilized PS003 was analyzed by a sandwich ELISA. In addition, as PS003 was selected on a recombinant form of PS, we analyzed the binding of a plasma-derived form of PS in solution to PS003 in the same sandwich ELISA. Briefly, rhPS and purified plasma-derived human PS (Haematologic Technologies) (60 μL at 10 μg/mL in TBS containing 5 mM CaCl.sub.2) were immobilized onto 96-well NUNC Maxisorp plates for 16 h at 4° C. Wells were washed with 3×200 4 of washing buffer (TBS containing 5 mM CaCl.sub.2) and 0.1% Tween-20) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2 and 5% BSA. Wells were washed with 3×200 μL of washing buffer and increasing concentrations (0-200 nM in TBS containing 5 mM CaCl.sub.2) and 1% BSA, 50 μL/well) of PS003 was incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-HA tag antibody (Abcam, 2 μg/mL in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature. Wells were washed with 3×200 4 of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. The results indicated that PS003 bound to either recombinant or plasma-derived human PS (
[0173] Comparison of PS003 and PS003biv Binding to Immobilized PS in ELISA
[0174] Recombinant human PS (rhPS) (60 μL at 2.5 μg/mL in TBS containing 5 mM CaCl.sub.2)) was immobilized onto 96-well NUNC Maxisorp plates, for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 5 mM CaCl.sub.2) and 0.1% Tween-20) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2) and 5% BSA. Wells were washed with 3×200 μL of washing buffer and increasing concentrations of PS003 and PS003biv (0-200 nM in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) were incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-His6 tag antibody (Abcam, 1 μg/mL in TBS containing 5 mM CaCl.sub.2) and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature to detect the bound nanobodies. Wells were washed with 3×200 4 of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. For each nanobody, three individual experiments were done in simplicate, and the results are expressed as percentage of maximal binding for each nanobody. Binding curves indicated that both PS003 and PS003biv efficiently bound to immobilized rhPS (
[0175] To further compare the ability of PS003 and PS003biv to bind to PS, the affinity of PS003 and PS003biv for rhPS was estimated as described (Beatty et al. J Immunol Methods 1987), by obtaining binding curves on rhPS immobilized at increasing concentrations (0.6, 1.25, 2.5 and 5 μg/mL in TBS containing 5 mM CaCl.sub.2) in three individual experiments done in simplicate. For each nanobody, the dissociation constant (K.sub.D) was determined by using a formula based on the Law of Mass Action.
[0176] Based on this method, the K.sub.D of PS003 and PS003biv were 26.8±2.7 nM and 13.8±5.7 nM, respectively, suggesting that PS003biv bound to rhPS with only slightly higher affinity (1.9-fold).
[0177] Epitope mapping of PS003biv and specificity of PS003biv for PS rhPS, a recombinant form of the PS SHBG-like region alone (rSHBG) (Saposnik et al. 2003), recombinant human Gas6 (rhGas6), or BSA (60 μL at 10 μg/mL in 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS) containing 5 mM CaCl.sub.2) were immobilized onto 96-well NUNC Maxisorp plates, for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 5 mM CaCl.sub.2 and 0.1% Tween-20) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2 and 5% BSA. Wells were washed with 3×200 μL of washing buffer and 0.5 nM PS003biv (in TBS containing 5 mM CaCl.sub.2, 0.1% Tween-20 and 2% BSA, 50 μL/well) were incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-His6 tag antibody (Abcam, 1 μg/mL in in TBS containing 5 mM CaCl.sub.2, 0.1% Tween-20 and 2% BSA, 50 μL/well) was incubated for 1 h at room temperature to detect the bound nanobodies. Wells were washed with 3×200 μL of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4. The results are expressed as the percentage of the Abs.sub.450 nm obtained on rhPS. Three individual experiments were done in simplicate.
[0178] The results indicated that PS003biv efficiently bound to rSBHG (
[0179] Enhancing Effects of PS003 and PS003biv on the APC-Cofactor Activity of PS in an APTT-Based Plasma Clotting Assay
[0180] We used a commercial APTT-based plasma clotting assay (STACLOT® PS, Stago) on a KC4 Coagulometer (Stago) to measure the ability of rhPS to act as a cofactor for APC in the inactivation of FVa and FVIIIa. Briefly, 25 μL of rhPS diluted in TBS containing 0.1% BSA were added to 25 μL of a PS-deficient plasma (R1 Reagent) together with APC (R2 Reagent, 4) and bovine FVa (Reagent R3, 25 μL). After a 2-min incubation at 37° C., clotting was triggered by the addition of 25 μL of 50 mM CaCl.sub.2. In this assay, APC prolonged the clotting times of the PS-deficient plasma, and rhPS (final concentration 5 nM) further prolonged, in a dose-dependent manner, the clotting times when rhPS was added together with APC (
[0181] A dose-response curve demonstrated that rhPS dose-dependently prolongs the clotting times in the presence of a fixed concentration of APC (
[0182] We then tested the effects of PS003 and PS003biv on the ability of rhPS (6 nM) to enhance the anticoagulant activity of APC. PS003, KB013, PS003biv, and KB004biv were pre-incubated at 10 μM with rhPS (30 nM) in TBS containing 0.1% BSA, for 15 min at room temperature. The mixture of rhPS ±nanobody (25 μL) was added to 25 μL of a PS-deficient plasma (R1 Reagent) together with APC (R2 Reagent, 25 μL) and bovine FVa (Reagent R3, 25 μL). After a 2-min incubation at 37° C., clotting was triggered by the addition of 25 μL of 50 mM CaCl.sub.2. The final concentrations of rhPS and the nananobodies were 6 nM and 2 μM, respectively. Experiments were done in triplicate.
[0183] In the presence of rhPS and APC, the clotting times were prolonged by approximately 2-fold in the absence (TBS) and in the presence of monovalent (KB013) and bivalent (KB004biv) nanobodies (final concentration 2 μM), which reflects the normal APC-cofactor of rhPS (
[0184] The previous results were expressed as ratios of clotting times in the presence of rhPS (t.sub.+PS) to the clotting times in the absence of rhPS (t.sub.−PS) (
[0185] Effects of PS003 and PS003biv on the APC-Cofactor Activity of PS in an In Vitro FVa Inactivation Assay
[0186] The capacity of PS003 and PS003biv to enhance the APC-cofactor activity of rhPS was evaluated in an in vitro assay measuring the specific proteolytic inactivation of FVa by APC, in the presence of rhPS, using purified proteins. Plasma-derived human FVa (Haematologic Technologies, 80 nM) was inactivated for 20 min with plasma-derived human APC (Haematologic Technologies, 0.5 nM), in the presence of 25 μM PC/PE/PS phospholipid vesicles and increasing concentration of rhPS (0-100 nM) in 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS) containing 5 mM CaCl.sub.2, 0.2% PEG and 0.2% BSA (“FVa inactivation mixture”). The reaction was stopped by diluting the FVa inactivation mixture (1:10) in TBS containing 5 mM CaCl.sub.2, 0.2% PEG and 0.2% BSA. The residual FVa activity was then measured in a prothrombinase assay with plasma-derived human prothrombin (Haematologic Technologies, 200 nM) and FXa (Enzyme Research Laboratories, 200 nM), in in TBS containing 5 mM CaCl.sub.2, 0.2% PEG and 0.2% BSA and 50 μM PC/PS/PE phospholipid vesicles. The amidolytic activity of thrombin was followed using a chromogenic substrate (pNAPEP0238, 200 μM) in TBS containing 10 mM EDTA, 0.2% PEG and 0.2% BSA, and the slope of progress curves were calculated. The slopes were determined for each rhPS concentration in the FVa inactivation mixture, and a value of FVa activity was expressed as the ratio between the slope obtained in the presence of rhPS and the slope obtained in the absence of rhPS. Three experiments were done in simplicate.
[0187] The results demonstrated that rhPS dose-dependently and very efficiently enhanced the ability of APC to inactivate FVa (
[0188] The results demonstrated that, in this APC-cofactor activity assay, the blocking anti-PS antibody (α-PS, DAKO) effectively inhibited the APC-cofactor activity of rhPS (
[0189] Effects of PS003 and PS003biv in an In Vitro TFPIα-Cofactor Activity Assay of PS
[0190] An in vitro assay has been developed to assess the ability of rhPS to enhance the direct inhibition of FXa by TFPIα. The amidolytic activity of plasma-derived human FXa (Enzyme Research Laboratories, final concentration 0.2 nM) towards a chromogenic substrate specific for FXa (pNAPEP, Cryopep, 400 μM) in a final volume of 100 μL of TBS containing 10 mM CaCl.sub.2, 0.2% PEG, 0.2% BSA and 25 μM PC/PS/PE phospholipid vesicles, was monitored every 8 s for 60 min. Recombinant human full-length TFPIα expressed in E. coli (kind gift by Tilman Hackeng, Maastricht, The Netherlands) was used at a final concentration of 5 nM to inhibit the amidolytic activity of FXa. We chose experimental conditions under which FXa was weakly inhibited by TFPIα alone but under which rhPS (final concentration, 20 nM) effectively enhanced FXa inhibition by TFPIα (
[0191] The ability of rhPS to enhance the inhibitory activity of TFPIα was abolished when rhPS was pre-incubated for 15 min at room temperature with a blocking rabbit polyclonal anti-PS antibody (α-PS) (DAKO, final concentration 0.5 μM), but not with rabbit IgG (DAKO, final concentration 0.5 μM (
[0192] The ability of rhPS to enhance the inhibitory activity of TFPIα was evaluated when rhPS was pre-incubated for 15 min at room temperature with PS003 and PS003biv, or their respective monovalent (KB013) and bivalent (KB004biv) control nanobodies (final concentration 10 μM). A kinetic constant (k.sub.obs) for the inhibition of FXa by TFPIα under each condition was calculated from the progress curves, as previously described (Ndonwi et al. 2010). The results were expressed as percentage of the TFPIα-cofactor activity of rhPS in the absence of nanobodies (TBS), and three experiments were done in simplicate, and Unpaired Student's t-test was used as a statistical test.
[0193] The results showed that PS003 and PS003biv did not potentiate, but rather slightly inhibited, the TFPIα-cofactor activity of rhPS, in this in vitro functional assay (
[0194] Binding of PS003biv and PS003biv to Immobilized Recombinant Murine PS (rmPS)
[0195] As for rhPS, recombinant murine PS (rmPS) was expressed in HEK293 cells in the presence of 10 μg/mL vitamin K1, and purified by a two-step anion-exchange chromatography, as previously described (Fernandez et al. 2009). rmPS (60 μL at 10 μg/mL in TBS containing 5 mM CaCl.sub.2) was immobilized onto 96-well NUNC Maxisorp plates, for 16 h at 4° C. Wells were washed with 3×200 μL of washing buffer (TBS containing 5 mM CaCl.sub.2) and 0.1% Tween-20) and wells were blocked for 1 h at room temperature with TBS containing 5 mM CaCl.sub.2 and 5% BSA. Wells were washed with 3×200 μL of washing buffer and increasing concentrations of PS003biv and PS004biv (0-50 nM in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) were incubated for 1 h at room temperature. Wells were washed with 3×200 μL of washing buffer, and a peroxidase-conjugated polyclonal anti-HA tag antibody (Abcam, 2 μg/mL in TBS containing 5 mM CaCl.sub.2 and 1% BSA, 50 μL/well) was incubated for 1 h at room temperature to detect the bound nanobodies. Wells were washed with 3×200 μL of washing buffer and 50 μL of TMB was added. Reactions were stopped by the addition of 50 μL of 2M H.sub.2SO.sub.4.
[0196] The results demonstrated that PS003biv strongly bound to rmPS (
[0197] As murine and human PS share 78% sequence homology between their SHBG-like regions, this result could also help localize the epitope for PS003biv. Indeed, candidate amino-acid residues are likely conserved within the SHBG-like region of human and murine PS, but not conserved within the the SHBG-like region of human Gas6.
[0198] In vivo antithrombotic effects of PS003biv in a mouse FeCl3-induced thrombosis model Ferric chloride (FeCl.sub.3)-injury was induced in 4- to 5-week-old C57BL6/JRccHsd male mice, essentially as previously described (Aymé et al. 2017; Adam et al. 2010). To facilitate visualization of thrombus formation, platelets of anesthetized mice with pentobarbital were fluorescently labeled in vivo by intravenous injection of rhodamine 6G (3.3 mg/kg, i.e. 2.5 μL/g of rhodamine 6G at 1 mg/mL in 0.9% NaCl) into the retro-orbital plexus. PS003biv (10 mg/kg), PS004biv (10 mg/kg), or the same volumes of TBS buffer (Ctl), were diluted in 0.9% NaCl and were administered simultaneously. Alternatively, 200 UI/kg low-molecular-weight heparin (LMWH, Lovenox) was injected subcutaneously after the intravenous injection of rhodamine 6G alone, to verify that our thrombosis model was sensitive to pharmacological inhibition of coagulation. The labelled platelets were left to circulate for 10 min, and after a topical deposition of FeCl.sub.3 solution (10% in water) on the mesenteric vessels, thrombus growth was monitored in real-time with an inverted epifluorescent microscope (×10) (Nikon Eclipse TE2000-U). One single venule and one single arteriole were analyzed for each mouse. Statistical analysis was assessed via Kruskal Wallis and Dunn's test. The results demonstrated that PS003biv exerted an antithrombotic effect in our FeCl.sub.3-induced thrombosis model in mouse mesenteric vessels. Treatment of mice with PS003biv resulted in delayed occlusion, especially in the veinules (
[0199] It should be noted that the control bivalent anti-VWF nanobody (KB004biv) used in our APC- and TFPIα-cofactor activity assays could not be used in our FeCl.sub.3-induced thrombosis model. Indeed, treatment of mice with this nanobody resulted in delayed occlusion times in the venule and arteriole of one mouse. Thus, we decided to use an in-house bivalent anti-PS nanobody (PS004biv) that is not able to bind to recombinant murine PS (
[0200] Effects of PS003biv on Physiological Hemostasis in a Mouse Tail-Clip Bleeding Model
[0201] Anesthetized C57/BL6 mice were intravenously injected with PS003biv (10 mg/kg) or subcutaneously injected with low-molecular-weight heparin (LMWH) (Lovenox, 200 UI/kg), as described for the FeCl.sub.3-induced thrombosis model. Tails were immerged at 37° C. for 10 min in 0.9% NaCl, and 3 mm from the tip of the tails were cut and immediately immerged at 37° C. into tubes containing 10 mL of 0.9% NaCl. Bleeding time was defined as the first cessation of bleeding. Blood was also collected during 20 minutes to quantify total blood loss volumes. Each bar represents the mean obtained from several mice evaluated. Ordinary one-way ANOVA was used as a statistical test of variance with Tukey's multiple comparison test.
[0202] The results demonstrated that this murine bleeding model was sensitive to pharmacological inhibition of coagulation, as administration of 200 UI/kg low-molecular-weight heparin (LMWH) significantly prolonged the bleeding times and significantly increased the blood loss volumes (
Discussion
[0203] We propose that PS003/PS003biv nanobodies might have a therapeutic interest in sickle cell disease (SCD). SCD is a genetic disease resulting from a point mutation in the HBB gene, leading to polymerization of hemoglobin S (HbS) during deoxygenation, and to deformation of red blood cells into a sickle shape. Such sickling impairs red blood cell transit in microvessels and renders them prone to hemolysis. Red blood cell lysis releases noxious mediators that among others, activate vascular endothelial cells and drive the adhesion of leukocytes and platelets to the activated endothelium. These pathological events ultimately result in microvascular obstructions leading to the recurrent and painful vaso-occlusive crisis (VOC) that characterize SCD. These vaso-occlusive events can eventually result in end-organ damage and, in many cases, premature death.
[0204] The pathophysiology of VOC is complex and involves an interplay between sickled red blood cells, endothelial cells, platelets, and leukocytes. In addition, SCD patients are generally thought to be in a chronic hypercoagulable state (Whelihan et al. JTH 2016), as evidenced by the elevated levels of thrombin-antithrombin complexes (TAT), prothrombin fragment F1.2 and D-dimers in these patients (Ataga et al. Hematology Am Soc Hematol Educ Program 2007). This hypercoagulable state is associated with an increased risk of venous thromboembolism and stroke that has been well described in SCD patients (Sparkenbaugh and Pawlinksi. JTH 2017; Brunson et al. Br J Haematol 2017; Shet et al. Blood 2018). However, chronic coagulation activation in SCD might also locally trigger and/or enhance vascular inflammation, which is a crucial pathophysiological feature of SCD. Indeed, it has long been recognized that coagulation and inflammation can amplify one to another in various thrombo-inflammatory diseases, and this crosstalk between coagulation and inflammation is believed to be central to the pathophysiology of vaso-occlusion in SCD (Sparkenbaugh et al. Br J Haematol 2013).
[0205] Tissue factor (TF) expression has been shown to be increased in leukocytes in SCD patients and in mouse models of SCD, suggesting that TF likely contributes to hypercoagulability in SCD. Leukocyte TF is considered as the most likely source of TF contributing to coagulation activation in SCD (Sparkenbaugh and Pawlinski. JTH 2017), but TF is also inducibly expressed in vascular endothelial cells. Little is known about the role of contact system in hypercoagulability in SCD. FXII might be activated at sites of phosphatidylserine exposure on various cell types (e.g. sickled red blood cells and endothelial cells) and microvesicles derived from endothelial cells, platelets or monocytes. This can be inferred from a study showing that FXII is able bind to phosphatidylserine exposed by apoptotic cells, leading to its rapid cleavage and activation (Yang et al. Front Immunol 2017). Such an activation of FXII by phosphatidylserine could be an additional trigger of coagulation activation in SCD, independently of TF. Alternatively, FXII and contact system could be activated by mast cell-derived products, such as glycosaminoglycans and heparin, or by glycated hemoglobin released by hemolysis (Sparkenbaugh and Pawlinski. JTH 2017).
[0206] Exposure of phosphatidylserine at the surface of sickled red blood cells, endothelial cells, and microvesicles is an important driver of hypercoagulability in SCD. This exposure of phosphatidylserine considerably accelerates the rates of coagulation reactions and might also enhance decryption and activation of TF (Ansari et al. Thromb Haemost 2019). Interestingly, PS has a high affinity for phosphatidylserine-containing anionic phospholipid membranes, suggesting that PS might accumulate at these sites and might exert an important anticoagulant role to locally limit thrombin generation. However, widespread vascular and intravascular exposure of phosphatidylserine could also lead to capture of PS and to depletion from its plasma pool. This would be in line with the apparent acquired deficiency in PS observed in SCD patients in various studies (Whelihan et al. JTH 2016). Acute or chronic hypoxia might also participate in decreased plasma levels of PS observed in SCD patients, as liver expression of PS was found to be downregulated by hypoxia through HIF-1α (Pilli et al. Blood 2018). How such PS deficiency contributes to hypercoagulability and exacerbates prothrombotic tendency in SCD patients is not known. Interestingly, protein C deficiency was also found in SCD patients (Whelihan et al. JTH 2016), suggesting that the anticoagulant protein C pathway might be more broadly altered in SCD. Indeed, combined deficiency in PS and protein C in SCD is expected to have a great impact on the ability of activated protein C (APC) to exert its anticoagulant activity. This is supported by the APC resistance found in the plasma of SCD patients, even though elevated FVIII levels in these patients might also be a contributing factor (Wright et al. 1997; Whelihan et al. 2016).
[0207] Importantly, local coagulation activation and generation of thrombin might directly play a major role in the pathophysiology of VOC, independently of its ability to generate fibrin during thrombus formation. Indeed, thrombin not only cleaves fibrinogen to generate fibrin but is also a potent activator of endothelial cells, platelets, and leukocytes, notably through PAR1 activation. In endothelial cells, thrombin exerts PAR1-dependent pro-inflammatory, pro-apoptotic, and barrier-disruptive effects in endothelial cells (Flaumenhaft and De Ceunynck. Trends Pharmacol Sci 2017). Furthermore, thrombin-mediated activation of PAR1 on endothelial cells induces exocytosis of Weibel-Palade bodies containing Willebrand factor (VWF) and P-selectin (Cleator et al. Blood 2014), which contributes to or enhances the interaction between sickled red blood cells and endothelial cells. In addition, such exocytosis of Weibel-Palade bodies could release other soluble mediators of vascular thrombo-inflammation, such as angiopoietin-2. Furthermore, thrombin could directly or indirectly induce the exposure of phosphatidylserine on endothelial cells, thus fueling and perpetuating coagulation and thrombin generation at their surface.
[0208] Consequently, local and low-grade generation of thrombin initiated by TF and the contact system at the endothelial surface might be an early trigger of vascular inflammation and vaso-occlusive events, even in the absence of thrombus formation and widespread coagulation activation. Very recently, an anti-TF antibody, direct oral anticoagulants targeting FXa (rivaroxaban) and thrombin (dabigatran), and a PAR1 antagonist (vorapaxar) all markedly reduced hemoglobin-induced microvascular stasis in a mouse model of VOC (Sparkenbaugh et al. Blood 2020). Thus, pharmacological targeting of thrombin-mediated endothelial PAR1 activation appears as an attractive therapeutic strategy to prevent and/or reduce VOC in SCD. This study also suggests that a proper control of thrombin generation at the endothelial surface by natural anticoagulants such as PS, APC and tissue factor pathway inhibitor (TFPI), might be crucial to limit VOC.
[0209] PS exhibits high affinity for phosphatidylserine exposed on the surface of activated endothelial surface, and has a unique ability to function as a cofactor for both APC and TFPI-α. By stimulating the anticoagulant activities of both APC and TFPI-α, PS might have a central role in the limitation of thrombin generation at the surface of endothelial cells in SCD. Consequently, PS could be an important negative regulator of thrombin-induced vaso-occlusive events, even though this has yet to be corroborated in experimental studies.
[0210] We here propose that enhancing the APC-cofactor activity of PS with PS003/PS003biv nanobodies might constitute a novel therapeutic strategy for preventing or reducing vaso-occlusive events in SCD patients. Through their antithrombotic properties, PS003/PS003biv could concomitantly help reduce the risk of venous thromboembolic events and stroke in treated patients, especially since PS deficiency and APC resistance are found in SCD patients.
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