Targeted Thrombolysis for Treatment of Microvascular Thrombosis
20210023187 ยท 2021-01-28
Assignee
Inventors
Cpc classification
C07K19/00
CHEMISTRY; METALLURGY
A61K38/16
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
C07K14/00
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides fusion proteins for targeted delivery of plasminogen activators to platelet-VWF complexes, or alternatively to the site where these are located, in a fibrin-independent manner. The fusion protein of the invention are for use in methods for the prevention or treatment of diseases or conditions associated with such platelet-VWF complexes, which may cause microvascular thrombosis in diseases such as e.g. thrombotic thrombocytopenic purpura. Preferred targeting agents for incorporation into the fusion proteins are e.g. nanobodies against VWF or platelets. Preferred plasminogen activators for use in the fusion proteins comprise the protease domains of uPA or tPA. The invention further pertains to nucleic acid molecule encoding the fusion proteins of the invention, e.g. a gene therapy vector, and to pharmaceutical compositions comprising the fusion proteins of the invention or such gene therapy vectors.
Claims
1.-9. (canceled)
10. A method for treating or reducing the risk of microvascular thrombosis, wherein the method comprises the step of administering to a subject in need thereof a fusion protein comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus comprising al least one of VWF and platelets, wherein the targeting agent is not a targeting agent that specifically binds to only the activated form of the GPIIb/IIIa receptor on platelets, for use in the prevention or treatment of microvascular thrombosis.
11. The method of claim 10, wherein the microvascular thrombosis is treated or the risk of its occurrence is reduced in a disease or condition selected from the group consisting of: acquired or hereditary thrombotic thrombocytopenic purpura (TTP) complement-mediated thrombotic microangiopathy, haemolytic uremic syndrome, antiphospholipid antibody syndrome, non-occlusive thrombus, the formation of an occlusive thrombus, arterial thrombus formation, acute coronary occlusion, peripheral arterial occlusive disease, restenosis and disorders arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting or atherectomy, hyperplasia after angioplasty, atherectomy or arterial stenting, occlusive syndrome in a vascular system or lack of patency of diseased arteries, transient cerebral ischemic attack, unstable or stable angina pectoris, cerebral infarction, HELLP syndrome, carotid endarterectomy, carotid artery stenosis, critical limb ischemia, cardioembolism, peripheral vascular disease, restenosis, sickle cell disease and myocardial infarct.
12. A fusion protein comprising a plasminogen activator and a targeting agent for targeting the plasminogen activator to a site of a thrombus comprising at least one of VWF and platelets, wherein the targeting agent is one or more of: a) a targeting agent that at least binds unfolded VWF, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF; b) a targeting agent that binds the D3 domain of VWF; c) a targeting agent that binds integrin allb/(3III on platelets; d) a targeting agent that binds to a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected form the group consisting of E-selectin, P-selectin, uPAR, c1q receptor, kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial protein C receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and, e) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatidylethanolamine.
13. A fusion protein according to claim 12, wherein the fusion protein comprises more than one targeting agent.
14. A fusion protein according to claim 12, wherein the targeting agent comprises at least one of: a) an antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and, b) a binding domain from a protein that naturally binds VWF, platelets and activated or injured vascular endothelium, which binding domain specifically binds to at least one of VWF, platelets, and activated or injured vascular endothelium.
15. A fusion protein according to claim 14, wherein the antibody variable domain is a VHH, preferably a humanized VHH, or wherein the binding domain comprises a binding domain selected from the group consisting of: i) a VWF-binding domain from one of ADAMTS13, Factor XII, Factor H (complement regulator), plasminogen and Factor VIII; and, ii) a membrane binding domain selected from the vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from factor V and the C-domain from factor VIII.
16. A fusion protein according to claim 12, wherein the plasminogen activator comprises the protease domain of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen, streptokinase or staphylokinase, wherein preferably the plasminogen activator further comprises at least the cysteine-containing part of the connecting peptide that naturally occurs in the plasminogen activator immediately upstream of its protease domain, and wherein the fusion protein optionally comprises a linker amino acid sequence linking the targeting agent and the plasminogen activator.
17. A fusion protein according to claim 16, wherein the fusion protein comprises in a N- to C-terminal order: a) one or more targeting agents wherein the targeting agent is one or more of: a targeting agent that at least hinds unfolded VWP, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF; ii) a targeting agent that binds the D3 domain of VWF; ill) a targeting agent that binds integrin IIb/III on platelets; iv) a targeting agent that binds io a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected form the groan consisting of E-selection, P-selection, uPAR, c1q receptor, kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial protein C receptor thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and, v) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatldylethanoiamine, whereby, optionally the targeting agents are linked by linker amino acid sequences; b) optionally a linker amino acid sequence; and, c) the plasminogen activator or plasminogen-derived protease domain.
18. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as defined claim 12, wherein the nucleotide sequence encoding the fusion protein further preferably comprises a nucleotide sequence encoding a signal peptide operably linked to the fusion protein, and wherein nucleic acid molecule further preferably comprises regulatory elements conducive to the expression of the fusion protein, which regulatory elements are operably linked to the nucleotide sequence.
19.-20. (canceled)
21. The method of claim 10, wherein the targeting agent specifically binds to at least one of VWF, platelets and activated or injured vascular endothelium.
22. The method of claim 10, wherein the targeting agent is one or more of; a) a targeting, agent that at least binds unfolded VWF, wherein preferably the targeting agent preferentially binds unfolded VWF over globular VWF. b) a targeting agent that binds the D3 domain of VWF; c) a targeting agent that binds the GP1B receptor on platelets, d) a targeting agent that binds integrin IIb/III on platelets; e) a targeting agent that binds to a receptor that is preferentially expressed by activated endothelium, wherein preferably the receptor is selected from the group consisting of E-setecan, P-selectin, uPAR, c1q receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and, f) a targeting agent that binds to a membrane marker for activated or injured endothelium, wherein the membrane marker is one or more of anionic phospholipids, phosphatidylserine and phosphatidylethanolamine.
23. The method of claim 10, wherein the fusion protein comprises more than one targeting agent.
24. The method of claim 10, wherein the targeting agent comprises at least one of: a) ao antibody variable domain that specifically binds to at least one of VWF, platelets, and activated vascular endothelium; and, b) a binding domain from a protein that naturally binds VWF, platelets and activated or injured vascular endothelium, which binding domain specifically binds to at least one of VWF, platelets, and activated or injured vascular endothelium.
25. The method of claim 24, wherein the antibody variable domain is a VHH, preferably a humanized VHH, or wherein the binding domain comprises a binding domain selected front the group consisting of: i) the platelet GP1B receptor-binding A1 domain from VWF; ii) a VWF-binding domain from one of ADAMTS13, Factor XII, Factor H (complement regulator), plasminogen and Factor VIII, and, iii) a membrane binding domain selected from the vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from factor V and the C-domain from factor VIII.
26. The method of claim 10, wherein the plasminogen activator comprises the protease domain of tissue plasminogen activator ((PAT urokinase plasminogen activator (nPA), plasminogen, streptokinase or staphylokinase, wherein preferably the plasminogen activator further comprises at least the cysteine-containing part of the connecting peptide that naturally occurs in the plasminogen activator immediately upstream of its protease domain, and wherein the fusion protein optionally comprises a linker amino acid sequence linking the targeting agent and the plasminogen activator
27. The method of claim 26. wherein the fusion protein comprises in a N- to C-terminal order; a) the one or more targeting agents, whereby, optionally the targeting agents axe linked by linker amino acid sequences; b) optionally a linker amino acid sequence; and, c) the plasminogen activator or plasminogen-derived protease domain.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1
Methods and Materials
[0118] Nanobody-mUPA construction
[0119] The cDNA sequence for both human and mouse urokinase (PLAU) was obtained from the NCBI database (NM_002658.4 and NM_008873.3 respectively). The sequence for the signal peptide, EGF-like and Kringle domain were removed as well as the first part of the connecting peptide. To the remaining connecting peptide and S1 peptidase domain (Catalytic domain) a N-terminal sequence coding for a Tobacco Etch Virus cleavage site followed by an GGGGS linker was added. In the GGGGS linked a Pstl and BamHI digestion site were incorporated without disturbing the amino acid sequence. At the 5 side an EcoRl digestion site was added and at the 3 side and Notl digestion was added after the STOP codon of PLAU. The construct was obtained from IDT (Integrated DNA Technologies, Leuven, Belgium) as a custom gene construct.
[0120] Coding sequences for nanobodies (also known as VHH) were codon optimized via IDT for expression in a human host cells. At the N-terminal side of the VHH coding sequence, a sequence coding for a Tobacco Etch Virus cleavage site was placed and at the C-terminal side a GGGGS linker (encoding a Pstl and BamHl digestion site). These DNA segments were obtained from IDT as Double stranded DNA fragments (gBLocks).
[0121] The custom gene construct was propagated in E.coli TOP10 and selected by ampicillin resistance. Obtained plasmid DNA was digested by EcoRl and Notl. The resulting insert (886) was separated on and isolated from agarose gel and ligated into a modified pcDNA6 expression vector (pSM2) (De Maat et al, 2016 J Allergy Clin Immunol Nov;30;138(5):1414-23)). pSM2 encodes a N-terminal murine IgK secretion signal and a double STREP isolation tag whereafter the modified UPA construct is ligated.
[0122] The gBlocks were ligated into the pJET1.2 cloning vector according to manufacturer instructions (CloneJET PCR Cloning Kit; Thermo Fisher). The constructs were propagated in E.coli TOP10 and selected by ampicillin resistance. Obtained plasmid DNA was digested by EcoRl and BamHl. The resulting insert was separated on and isolated from agarose gel and ligated into the pSM2 vector containing the miniUPA construct. The gene constructs are schematically depicted in in
TABLE-US-00004 TABLE 1 Description of nanobody-mUPA fusion protein constructs Specificity Name Target VWF sVWF-mUPA globular and unfolded VWF D3-mUPA D3 domain of VWF A11-mUPA A1 domain of VWF A12-mUPA A1 domain of VWF Platelets GP1B17-mUPA platelet GP1B receptor Negative control R2-mUPA no binding to VWF or platelets
Nanobody-mUPA Production
[0123] The nanobody/mUPA-pSM2 constructs were transfected into HEK293 FreeStyle cells using 239Fectin as instructed by the manufacturer (ThermoFisher). After 1 day cells were expanded to a 20 mL. 2 Days hereafter the cells were placed under blasticidin (5 g/mL) selection. Transfected cells were further cultured according to manufacturer instructions until the constructs were stably integrated into the HEK genome. Cell were expanded (to 1.1*10.sup.6 cells/mL) and after 7 days of protein production, the cells were spun down at 2000g for 5 minutes. Hereafter the supernatant was collected and benzamine (0.174 mg/mL) was added, where after the supernatant was stored at -20 C. until further use.
[0124] Nanobody-mUPA purification
[0125] Collected supernatant (400 mL) was concentrated on a Quixstand to 150 mL using a 10 kDA cutoff membrane (GE healthcare). Hereafter the concentrate was dialyzed against 2L of 1 STREP buffer containing benzamidine (100 mM Tris, 150 mM NaCl, 0.174 mg/mL Benzamidine, pH 8.0). The concentrate was flowed over a column containing 8 mL Strep-Tactin superflow beads (IBA). After washing the column with 20 mL of 1 STREP buffer, the protein was eluted via d-Desthiobiotin (2.5 mM; Sigma Aldrich) in 1 STREP buffer. The purified proteins were dialyzed against 2L sodium acetate (4 mM sodium acetate, 150 mM NaCl, pH 5.4) and stored at 80 C.
Results
Purified Nanobody-mUPA Constructs on Western Blot
[0126] The nanobody-mUPA constructs were diluted in sample buffer (25 mM DTT) to a concentration of 100 g/mL. 10 L sample was loaded onto a 4-12% gradient Bis-Tris gel in MES buffer. Sample separation was performed at 165 Volt for 50 minutes. The gel is transferred onto a Immobilon-FL membrane in blotting buffer for 1 hour at 125 Volt. The membrane is blocked with 0.5 Odyssey blocking buffer where after the constructs are detected with a rabbit polyclonal anti human UPA antibody in combination with an 1R800 labeled Goat-anti-Rabbit antibody. Results were analyzed via the near-infrared odyssey scanner (Licor) according to manufacturer instructions.
Urokinase Activity of Nanobody-mUPA Constructs
[0127] The nanobody-mUPA constructs should display no spontaneous (i.e. non-induced) activity but should be activatable through molecular cleavage by plasmin.
[0128] To verify that none of the nanobody-mUPA constructs shows any spontaneous activity, the constructs (1 g/mL) were incubated in 0.2% BSA-HBS in the presence of a 0.5 mM urokinase substrate (11140; Bachem). Substrate conversion was measured according to manufacturer instruction at 37 C. None of the fusion proteins showed any detectable spontaneous activity towards the urokinase substrate (data not shown).
[0129] To test whether nanobody-mUPA constructs are activatable by plasmin, plasminogen was pre-activated by streptokinase for 15 minutes at 37 C. (hereafter called plasmin). The nanobody-mUPA fusion constructs (1 g/mL final concentration) were diluted in in 0.2% BSA-HBS and incubated for 12 minutes with plasmin (1 g/mL final concentration). Subsequently, urokinase substrate (0.5 mM 11140, Bachem) was added and its conversion was measured according to manufacturer's instructions at 37 C.
Plasminogen Activation
[0130] The basis of plasminogen activation by urokinase depends on the reciprocal cleavage between urokinase and plasminogen. To test plasminogen activation by the nanobody-mUPA constructs, the constructs (1 g/mL) were incubated in 0.2% BSA-HBS in the presence of 100 g/mL plasminogen and 0.2 mM plasmin substrate (11390; Bachem). Substrate conversion was measured according to manufacturer instruction at 37 C.
Binding of Anti-VWF Constructs to VWF as Determined by ELISA
[0131] A nunc maxisorp plate (Thermo) was coated overnight with 1 g/mL VWF in PBS. The following day the plate was blocked with 1% BSA-PBS. The anti-VWF nanobody-mUPA fusion constructs were diluted in 1% BSA-PBS at various concentrations after which 50 uL is added to each well and incubated for 1 hours. Hereafter the plate is washed with PBS-Tween 20 (PBST; 0.05% v/v). Bound construct was detected via rabbit anti-UPA polyclonal antibody in combination Goat-anti-rabbit-HRP secondary antibody (Abcam). Wells were rinsed with PBST, after which 100 l TMB was added at room temperature. Substrate was developed for 5 minutes after which 50 l H.sub.2SO.sub.4 (0.3 M) was added. Results were analyzed at 450 nm by absorption. Results were analyzed by Graphpad Prism 7.02 and the K.sub.d (in nM) was determined via non-linear regression curve fit. Binding affinities are listed in Table 2.
TABLE-US-00005 TABLE 2 Binding affinities of anti-VWF nanobody-mUPA fusion constructs to VWF as determined by ELISA. Construct K.sub.d (nM) Standard deviation sVWF-mUPA 0.03055 0.02491 D3-mUPA 0.3279 0.2005 A12-mUPA 12.84 6.571 R2-mUPA 1039010 1798731
Plasminogen Activation in the Presence of Globular or Open VWF
[0132] VWF is the scaffold for microthrombus formation. It also has plasmin(ogen) binding properties that are dependent on protein conformation (Tersteeg et al., 2014, supra). VWF unfolds under shear stress (as well as during immobilization on microtiter plates see previous experiment). This can be mimicked by incubation with the small molecule ristocetin. We asked whether plasminogen activation by our targeted fusion proteins is influenced by the conformation of VWF. The nanobody fusion constructs (0.25 g/mL) and VWF (5 g/mL) are diluted in 0.2% BSA-HBS. Hereafter, ristocetin (0.6 mg/mL) or buffer is added to open up the VWF or keep it globular, respectively, while incubating at 37 C. for 5 minutes. Hereafter plasminogen is added (100 g/mL) followed by plasmin substrate (11390 Bachem; 0.2 mM). Substrate conversion was measured according to manufacturer's instructions at 37 C. For comparison, the substrate at conversion after 5 minutes was shown for the different constructs. In the bar graph below. Data was processed in Graphpad Prism 7.02 and analyzed by one-way Anova. * P<0.05.
Micro-Thrombolysis of VWF-Platelet Agglutinates.
[0133] Blood platelets were isolated from citrated whole blood according to earlier described methods (Tersteeg et al., 2014, supra). Isolated blood platelets (200.000 /mL) were incubated with VWF (5 g/mL), plasminogen (100 g/mL) and the aggregation inhibitors RGDW (200 M) and Iloprost (0.4 g/mL) for 15 minutes at 37 C. in a light transmission aggregometer. Agglutination was induced by the addition of ristocetin (0.6 mg/mL). 6 minutes hereafter, the nanobody-mUPA constructs (1 g/mL) were added and lysis of the agglutinates was monitored overtime (
Example 2
Micro-Thrombolysis of VWF-Platelet on Endothelial Cells in Flow Perfusion Materials and Methods
Human Vascular Endothelial Cell (Huvecs) Culture on Cover Glasses
[0134] Huvecs (passage 0) stored in liquid nitrogen were thawed at 37 C. and added to medium 1:10 (EBM-2 Lonza or Promocel supplemented with huvec growth factors EGM2) and spun down 100 g for 5 minutes. Supernatant was discarded and cells were taken up in 5 mL medium and cultured in T25 flasks at 37 degrees Celsius, 5% CO.sub.2. The following day the cells were passed to 3 T75 flasks. On day 6 the cells are passed 1:6 to the cover glasses pre-treated with 1,25% glutaraldehyde in HT-buffer pH 7.4 (HEPES Tyrode buffer: 10 mM HEPES, 0.5 mM Na.sub.2HPO4, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4).
[0135] To coat cover glasses with glutaraldehyde, coverglasses were rinsed with demi water and with ethanol. Cover glasses were then incubated in HCL 37% : methanol (1:1) for 30 minutes followed by a rinse with demi water for 5 minutes. Next cover glasses were incubated in aminopropyltriethoxysilane : ethanol (1:100) for 30 seconds followed by a rinse with demi water and with ethanol. Cover glasses were then dried and incubated with glutaraldehyde 20% : HT-buffer pH 7.4 (1:20) for 1 hour followed by a rinse with demi water and glasses are stored in ethanol until use.
[0136] Huvecs were cultured on the cover glasses for approximately 10-15 days prior to use.
Heat Inactivated Plasma Preparation
[0137] Heat inactivated plasma was prepared by mixing two bags of plasma (Ominplasma from octapharma both bloodtype AB Lot No: C442A9521, bags: X000214223782; X000214223577). 200 mL of this mix was divided over 10 falcon tubes (50mL, 20mL each) and were incubated in a water bath set at exactly 56 C. for 30 minutes (the 20 mL was completely submerged) and halfway through (15 min) the falcon tubes were mixed. Following the 30 min incubation the falcon tubes were covered in ice and kept on ice until centrifuging (note: when removing the tubes from ice for centrifugation the tubes were still rather warm). The tubes were centrifuged 5 minutes (without cooling) at 15.000 g. The supernatant was combined and kept on ice until aliquoting in 1 mL aliquots.
Flow Chamber Setup
[0138] A laminar-flow perfusion chamber was filled with pre-warmed medium to remove all air from the tubing prior to placing the coverslips. The inlet tube was cut to a length that corresponds with a volume of 90,6 L so that during the perfusion the fluid that enters the inlet arrives into the perfusion chamber after exactly 1 minute. The syringe that is used has a diameter of 16 mm (only for braun 12 ml syringes) and the syringe pump is set to 90,6 L per minute as this (with 3 mm tubing) results in a shear rate of 300 s.sup.1. The huvec coverslip is placed on the medium, attached with the vacuum set at 10 bar and the perfusion chamber is placed under an inverted microscope (Zeiss observer Z.1, Carl Zeiss) with heating module that keeps the perfusion chamber at 37 C. Any remaining air bubbles are removed by perfusing medium.
Washed Platelets in Heat Inactivated Plasma
[0139] Blood from healthy consenting volunteers was collected into 0.1 volume 3.2% 10.9 mM trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation (160 g for 15 min at RT). PRP supplemented with 10% (v/v) acid citrate dextrose, 85 mM tri-sodiumcitrate, 71 mM citric acid, 111 mM D-glucose, was centrifuged (400 g for 15 min at RT) and platelets were resuspended in HEPES tyrode buffer pH 6.5 containing 0.145M NaCl, 5mM KCl, 0.5mM Na.sub.2HPO.sub.4, 1mM MgSO.sub.4, 10mM HEPES and 5.5 mM D-glucose. 10 g/mL PGl.sub.2 was added to the platelet suspension prior to another centrifugation step (400 g for 15 min at room temperature) and platelets were resuspended in heat inactivated plasma and platelet count was adjusted to a final count of 200 G/L.
Perfusion Experiment Setup
[0140] The heat inactivated plasma containing 200 G/L platelets (that is pre-warmed in 37 C. waterbath) is divided over 2 mL eppendorfcups (40 min experiment.fwdarw.22mL required, etc). and prior to the start to all eppendorfcups illoprost (8 L (250 fold dilution) of 0.1 mg/mL stock is added, final concentration 0.4 g/mL, Bayer Schering Pharma AG) is added first, followed by histamine (4 L (500 fold dilution) of 500 M stock in medium, final concentration 100 M). The perfusion is started immediately after the addition of Iloprost and histamine by transferring the inlet tube from the 2 mL eppendorfcup containing medium to the eppendorfcup containing heat inactivated plasma with platelets by squeezing the tube to ensure no air is introduced. The experiment is started with frame 1 after approximately 1 minute, when the first platelets enter the flow chamber (visible change). After 7 minutes the constructs are added to the remaining (2000-634.2=1365.8 L because 7*90.6=634 L is used at this point) by pipetting the required volume directly into the eppendorfcup and mixing with a plastic pipette, final concentration is 10 g/mL. At this point the construct is also added to the additional Eppendorf cups to make sure the pre-incubation time with plasma remains equal. During the perfusion the 2mL Eppendorf cup needs to be re-filled by carefully adding plasma with a plastic pipette drop by drop. This refill is done at frames 150, 250, etc. so that potential disturbances due to the refilling can be matched to the corresponding frames/time points. During the experiment every 5 seconds a DIC image is made and at the end of the experiment 5 screenshots are made of other regions in the perfusion chamber and the number of platelet-VWF complexes, visible in the form of platelet-strings are counted.
Results
[0141] The results are shown in
Example 3
Methods and Materials
Caplacizumab Production
Cloning
[0142] Caplacizumab (a bi-valent variant of the Cablivi VHH) was produced in E.coli and purified via HIS-tag affinity chromatography. The Caplacizumab protein sequence was derived from its EMA assessment report (EMA/490172/2018; Procedure No. EMEA/H/C/004426/0000) and codon optimized for E.coli expression via the integrated DNA technologies (IDT) codon optimized tool. N-terminal BamHl and C-terminal Notl digestion sites were added to the constructs and ordered as a double stranded DNA fragment from IDT (SEQ ID NO:40).
[0143] The DNA fragment were dissolved in 5 mM Tris-buffer (pH=8.5) and heated at 50 C. for 20 minutes. Hereafter the DNA fragment was ligated into the pJET1.2 vector (CloneJET PCR Cloning Kit; Thermo Fisher) according to manufacturer instructions. The ligated product was transformed into chemically competent E.coli TOP10 (Thermo Fisher) via heat shock according to manufacturer instructions. Transformed bacteria were cultured in 10 mL 2YT media (containing 100 g/mL ampicillin) and grown overnight at 37 C. Plasmid DNA was isolated via the plasmid isolation kit (M&N) according to manufacturer instructions. The insert was digested via BamHl-HF and Notl-HF (NEB) in Cutsmart buffer and separated on 0.7% (w/v) agarose gel (1TBE buffer; 1:10.00 gel red) at 130V for 1 hours. The insert was excised from gel and purified via the PCR&Gel cleanup kit (M&N) according to manufacturer instructions.
[0144] The purified insert was ligated into the pTH4.0 vector. The pTH4.0 vector is a modified pET32a(+) vector encoding an N-terminal PeIB signal peptide; a His6 tag for purification purposes and a sequence encoding a cleavage site for the tobacco etch virus (TEV) protease followed by a BamHl digestion site. After the C-terminal Notl digestion a myc-tag was placed for detection purposes followed by a stop-codon (Table 1). The pTH4.0 vector was digested via BamHI-HF and Notl-HF and subsequently purified as described for the fragments. The fragment were ligated into the digested pTH4.0 vector in a 3:1 ratio via T4 ligase in 1 T4 ligate buffer according to manufacturer instructions. The ligation mixture was transformed in TOP10 bacteria as described before, and the transformed bacteria were grown on YT-agar plate (100 g/mL ampicillin, 2% (w/v) glucose) overnight at 37 C. Colonies were picked, and grown in 10 mL 2YT media (containing 100 g/mL ampicillin, 2% (w/v) glucose). Plasmid DNA was isolated as described before. The DNA sequence was confirmed by sanger-sequencing via Macrogen (SEQ ID NO: 41).
Production
[0145] Caplacizumab in pTH4.0 plasmid DNA was transformed into chemically competent BL21 pLysS E.coli (Thermo) according to manufacturer instructions. Transformed bacteria were cultured overnight at 37 C. in 10 mL 2YT media (containing 100 g/mL ampicillin, 34 g/mL chloramphenicol and 2% (w/v) glucose). The overnight was diluted 1:10 in 2YT media (containing 100 g/mL ampicillin, 34 g/mL chloramphenicol and 2% (w/v) glucose) and grown for 3 hours at 37 C. Hereafter the culture was diluted 1:100 in 2YT media (containing 100 g/mL ampicillin, 34 g/mL chloramphenicol) and grown at 37 C. for 3 hours. When the bacteria reached OD600nm=0.6, protein production was induced by the addition of Isopropyl -D-1-thiogalactopyranoside (0.1 mM final concentration). Protein production was performed overnight at 24 C. Bacteria were pelleted at 5000g for 15 minutes and the supernatant was discarded. The bacteria pellet from 400 ml culture was resuspended in 25 mL dulbecco's phosphate buffer saline (PBS; 137 mM NaCl, 2.7mM KCl, 1.5 mM KH2PO4, 8.2 mM Na2HPO4, pH=7.4) and frozen at 20 C.
Purification
[0146] The frozen bacteria were thawed at 37 C. and pelleted at 10.000g for 15 min by centrifugation. The supernatant was transferred to new tubes. 5 mL of Cobalt-sepharose beads (TALON Superflow, G&E Heatlhcare; 50% solution;) were washed by PBS according to manufacturer instructions, added to the supernatant and incubated for 2 hours at room temperature on a roller bench. The TALON was pelleted at 1000g for 5 minutes by centrifugation. Supernatant was discarded and the pellets were dissolved in 20 mL of PBS. The TALON washing was repeated three times in total. After the last step, the TALON was dissolved in 8 mL of PBS and loaded into a PD-10 column. The column was rinsed with an excess of PBS. The column was eluted with Imidazole (150 mM in PBS) and 0.5 mL fractions were collected. Protein containing fraction were pooled and dialyzed overnight against HEPES-buffered saline (HBS: 10mM HEPES, 150 mM NaCl, pH=7.4) via a 3.500 MWCO dialysis membrane (3 RC tubing; Spectra/Por). Protein concentration was determined by absorption at 280 nm on the DeNovix Spectrophotometer (DS-11), where after the concentrations were corrected for their extinction coefficient (calculated via ProtParam). Purity was assessed by SDS-PAGE with Coomassie Page Blue staining.
Microthrombolysis of VWF-Platelet Agglutinates.
[0147] Blood platelets were isolated from citrated whole blood according to earlier described methods (Tersteeg et al., 2014, supra). Isolated blood platelets (200.000 /L) were incubated with VWF (5 g/mL), plasma-purified plasminogen (100 g/mL) and the aggregation inhibitors RGDW (200 M) and Iloprost (0.4 g/mL) for 15 minutes at 37 C. in a light transmission aggregometer. Agglutination was induced by the addition of ristocetin (0.6 mg/mL). 6 minutes hereafter, the nanobody-mUPA constructs or Caplacizumab were added and lysis of the agglutinates was monitored overtime. For all samples, time points were determined by which 50% microthrombus degradation has occurred (graphic example of analytical method is shown in
Results
[0148] Results are shown in
TABLE-US-00006 TABLE3 Descriptionofthesequences SEQIDNO: Seqname Sequence 1 hmUPA LKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLIS PCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSA DTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFG KENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKT DSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPW IRSHTKEENGLAL 2 mmUPA QGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKCGGS LISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLILHEY YREDSLAYHNDIALLKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEITGF GKESESDYLYPKNLKMSVVKLVSHEQCMQPHYYGSEINYKMLCAADPEW KTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFLD WIQSHIGEEKGLAF 3 secretionsignal METDTLLLWVLLLWVPGSTGD peptide 4 2xSTREP GSSAWSHPQFEKGSSAWSHPQFEK 5 TEV EFENLYFQS 6 LINKER SAAGGGGSGGGGSAAA 7 sVWF-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAMGWFRQAPGKE REFVAAISWSGGSTYYLDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CAGSAGLGYVGDPDAMDYWGKGTQVTVSSSAAGGGGSGGGGSAAALK FQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 8 D3-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGQTLSNYVMGWFRQAPGKE REFVAVISRVGGSTSYADSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CAAAYTIAVVTAMREYDFWGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 9 R2-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSQVQLQESGGGLVQAGGSLRLSCAASGRATSGHGHYGMGWFRQV PGKEREFVAAIRWSGKETWYKDSVKGRFTISRDNAKTTVYLQMNSLKPED TAVYYCAARPVRVDDISLPVGFDYWGQGTQVTVSSSAAGGGGSGGGGSA AALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSL ISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL 10 A11-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKER EFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCA ARSSAFSSGIYYREGSYAYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 11 A12-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSQVQLVESGGGLVQAGGSLRLSCTASGRTFSTYALGWFRQVPGKGR EFIAVIYWRDGSSLYSDSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYC ANRHDSRGTYYSSRGYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 12 GP1B-17hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASDIFSINAMGWYRQAPGKQRE LVASITRGGDPWYADSVKGRFTISRDGAKNARNTVYLQMNSLKPEDTAVY YCNAMGIRGSGGDYAREAGGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 13 12A5 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVAT ITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQ GSYGYRFNDYWGQGTQVTVSS 14 12A5H1 EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVAT ITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLRAEDTAVYYCYANLKQ GSYGYRFNDYWGQGTQVTVSS 15 R2-mmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSQVQLQESGGGLVQAGGSLRLSCAASGRATSGHGHYGMGWFRQV PGKEREFVAAIRWSGKETWYKDSVKGRFTISRDNAKTTVYLQMNSLKPED TAVYYCAARPVRVDDISLPVGFDYWGQGTQVTVSSSAAGGGGSGGGGSA AAQGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKC GGSLISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLIL HEYYREDSLAYHNDIALLKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEI TGFGKESESDYLYPKNLKMSVVKLVSHEQCMQPHYYGSEINYKMLCAADP EWKTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFL DWIQSHIGEEKGLAF 16 uPAconnecting ADGKKPSSPPEELKFQCGQKTLRPRFK peptide 17 tPAconnecting STCGLRQYSQPQFR peptide 18 Plasmin PSFDCGKPQVEPKKCPGR connecting peptide 19 tPAcalatytic STCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILIS domainsequence SCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDD DTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGK HEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVT NYLDWIRDNMRP 20 GPB1-1-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKG LEWVSAINTGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYY CAKDLPNSDSLGYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY LYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGD SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 21 GPB1-2-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLICVGSDRIFSNYSMGWFRQAPGKE RQFVSTISRHGTSTAYADSVRGRFTISRDNAENIVYLQMNSLEPEDTAVYYC AARPHTQHYVRVESYGVWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 22 GPB1-3-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGSINSIRAMGWYRQPPGKQR ELVATITRDGRTNYPDSVKGQFTISIDNARNTVSLQRNSLKPEDTAVYYCVA DWGEGYLTRVWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTL RPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHC FIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIA LLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPE QLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGG PLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENG LAL 23 GPB1-4hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASETFSIRAMGWYRQAPGKQRE LVAYITSGGSTNYADSVKGRFTISRDNDRNTVSLQMNSLKPEDTAVYYCYQ APRSGYDPVYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTLR PRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFI DYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALL KIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQL KMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLV CSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL 24 GPB1-5hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASEFTFSKHWMYWVRQAPGKG LEWVSGINLGGDSTYYADSVKGRFTISRDNAKNTLYLQMDSLKSEDTAVYY CAKGASSWFGDFGSWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY LYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGD SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 25 GPB1-6hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFAMNWVRQAPGKG LEWVSFINRGGGSTGYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVY YCAKFSRSVPPYYGMDYWGKGTLVTVSSSAAGGGGSGGGGSAAALKFQC GQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVI SATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVITKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 26 GPB1-7hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRVDSMAWFRQAPGKEREF VATITWSDSKIYYADSVKGRFTISGERAKNTMYLQMNTLRPEDTAVYYCAA AHRPYRSGYYYMQSRYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 27 GPB1-8hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAAPSMFSINAMGWYRQAPGRQR ELVATITSGDSTYYADSVKGRFTISRDNAKYTKNTVYLQMNSLKPEDTAVYY CNAAHIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 28 GPB1-9hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGPTVSNYYMGWFRQAPGKE RDFVAGISRSGVEKYYADSVKGRFTISRDNALNTVYLQMNSLKPEDTAAYY CAARERVGITFAHSTVDYWGKGTLVTVSSSAAGGGGSGGGGSAAALKFQ CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 29 GPB1-10hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSKYGMSWVRQAPGKGL EWVSIIDSGGGAIGYADAVKGRFTISRDNVKNTLYLQMNSLKPEDTAVYHC VFGDYKGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTLRPRFKIIG GEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKE DYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKE GRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTV VKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQ GRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL 30 GPB1-11hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMTWVRQAPGKGL EWVSAINSGGSGTRYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYY CAKRRDGQNWYPGISYESMYRGQGTQVTVSSSAAGGGGSGGGGSAAAL KFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISP CWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSAD TLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 31 GPB1-12hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYTMAWFRQAPGKER EFVGLISWNAKSTYVTDSVKGRFTITRENAKDMVYLQMNSLKPEDSATYYC AANRYGSSVPGAYNYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY LYPEQLKMTVVKLISHRECQQPHYYGSEVITKMLCAADPQWKTDSCQGD SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 32 GPB1-13hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGL EWVSAINMGGGSTYYADSVKGRFTISRDNAKNTLYLQMSGLKPEDTALYY CVRGGSAYSVRYEYAYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQC GQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVI SATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 33 GPB1-14hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAAAASWFSIYAMGWYRQAPGKQ RELVAIILSDGDTDYADSVKGRFTISRDNAKNTKNTVYLQMNSLKPEDTAV YYCNARGIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAALK FQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 34 GPB1-15hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGSMFSINDMGWYRQAPGK QRELVATITRGGNTYYADSVKGRFTISRDNATYTKNTVYLQMNSLKPEDTA VYYCNARHIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAAL KFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISP CWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSAD TLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 35 GPB1-16hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASRRTFSNYVMGWFRQAPGKE RESVTAIGRSGTILYADSMKGRITISRDNAKNTVYLQMNSLTPDDTAVYYC AASSGSMQQFWRMEYDYEGQGTQVTVSSSAAGGGGSGGGGSAAALKF QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 36 GPB1-19hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFGSYVMGWFRQAPGKE REFVAAIGRSGTTYYLDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYC GASLKGTVLGIARYEYDVRGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 37 GPB1-20hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGSVQAGGSLRLSCAASGRTLSSLAMGWFRQAPGKER EFVAADRRNGGYTVVADYTDSVKGRFTIFRDNAKNTVYLQMNNLKPEDT AVYYCAADSDRTMSLRSTDYDYWGQGTQVIVSSSAAGGGGSGGGGSAA ALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLI SPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL 38 Flexiblelinker KESGSVSSEQLAQFRSLD 39 Flexiblelinker EGKSSGSGSESKST 40 Caplacizumab GGATCCGAAGTCCAGCTTGTAGAATCAGGAGGAGGCCTTGTCCAGCCA doublestranded GGTGGTAGCCTTCGTCTGTCGTGTGCTGCTTCGGGCCGCACATTTTCGT DNAfragment ATAACCCTATGGGTTGGTTTCGCCAAGCACCCGGCAAGGGACGCGAGT TGGTGGCCGCGATTAGTCGTACGGGTGGTTCCACCTACTACCCGGATTC AGTGGAAGGACGCTTTACGATTAGCCGTGATAACGCGAAGCGTATGGT CTACTTACAGATGAATAGCTTGCGCGCGGAAGACACCGCGGTATACTA TTGTGCTGCAGCAGGAGTCCGTGCTGAGGATGGACGCGTCCGCACGTT ACCTAGTGAGTATACATTCTGGGGCCAGGGCACCCAAGTTACCGTATCC AGTGCAGCAGCGGAAGTACAACTGGTCGAATCTGGAGGAGGACTTGT ACAACCAGGGGGTTCCTTACGTTTGTCATGTGCGGCAAGTGGGCGCAC ATTTAGTTACAACCCTATGGGCTGGTTCCGTCAAGCCCCGGGAAAAGG GCGCGAACTTGTAGCCGCCATTTCGCGTACAGGGGGAAGTACCTATTA CCCGGACTCAGTAGAGGGACGCTTCACGATTTCTCGTGACAACGCAAA GCGCATGGTTTATCTGCAAATGAATAGTTTACGCGCCGAAGATACAGC AGTTTACTATTGCGCCGCAGCTGGAGTCCGCGCCGAAGACGGCCGTGT ACGCACCTTGCCTTCTGAATACACTTTTTGGGGTCAAGGAACACAGGTG ACCGTGTCATCTGCGGCCGC 41 Caplacizumabin MKYLLPTAAAGLLLLAAQPAMAQSGHHHHHHHHDYDIPSSENLYFQGSE pTH4.0protein VQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAA sequence ISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAG VRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSL RLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRF TISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSSAAAEQKLISEEDL 42 PrimerFw TAATACGACTCACTATAGGG 43 PrimerRv GCTAGTTATTGCTCAGCGG 44 Cablivi-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKG RELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY CAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSSAAGGGGSGGGGSAA ALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLI SPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL 45 Cablivi-mmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN LYFQSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKG RELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY CAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSSAAGGGGSGGGGSAA AQGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKCG GSLISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLIL 46 Cablivi EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA GVRAEDGRVRTLPSEYTFWGQGTQVTVSS