Final Report Summary - DIREKT (Disarming the intravascular innate immune response to improve treatment modalities for chronic kidney disease)
Executive Summary:
Chronic kidney disease is a major cause of end-stage renal disease (ESRD) worldwide: 800,000 patients in Europe and the US require long-term treatment, initially with peritoneal dialysis, and then with haemodialysis and kidney transplantation. An ESRD patient on haemodialysis has poor quality of life, with an average life expectancy of only 4 years and healthcare costs of ≈€40,000 to €80,000 per year. Kidney transplantation totally changes life for an ESRD patient, who can then return to normal life, but more widespread adoption of this treatment is still hampered by the low number of available kidney grafts. All these treatments are associated with adverse reactions that cause damaging thromboinflammation. These reactions are triggered by the intravascular innate immune system, leading to poor results and non-function.
The overall aim of this project was to clarify the mechanisms and identify the specific control points of regulation in these adverse reactions. In order to be able to significantly improve the quality of haemodialysis devices and kidney grafts, we have developed new concepts of regulation in haemodialysis and kidney transplantation. The results of this research and development have been applied in three distinctive clinical treatment modalities and in assays for the analysis of thromboinflammation in animal models:
1. We have brought a novel soluble complement inhibitor from prototype drug to a completed phase 1 clinical trial.
2. We have created nano-profiled surfaces with low activating properties that have been applied to catheters intended for haemodialysis use.
3. We have developed a PEG-lipid construct which can be used to coat biomaterial surfaces and have used it ex-vivo to coat kidney grafts intravascularly before transplantation, significantly protecting the kidney grafts against ischemia/reperfusion injury.
4. Finally, we have developed eight assays for the assessment of thromboinflammation in the mouse and pig for use in models of human disease and conditions in which thromboinflammation contributes to the pathophysiology.
All three treatment modalities are at varying stages of development into clinical treatment regimens for ESRD. When applied, we anticipate that these modalities will extend the feasible hemodialysis treatment period and, in kidney transplantation, attenuate innate immune reactions, thereby prolonging the life expectancy of the grafts and making more kidneys accessible for transplantation. The impact of these innovations is an improved quality of life and lowered cost of treatment for ESRD patients. Furthermore, all these novel techniques can be applied to other types of implantation, extracorporeal treatment, and organ transplantation and can potentially be used in the future in xenotransplantation and stem cell therapies.
Project Context and Objectives:
1. Introduction
Innate immunity is fundamental to our defense against microorganisms and foreign substances and controls the discrimination between self and non-self structures in the human body. As a consequence of its properties and actions, it is responsible for many of the incompatibility reactions that occur when foreign substances, materials, cells, and organs are introduced into the body. These reactions pose a major problem when modern biotechnological treatment modalities are used, including biomaterial devices, drug delivery systems, various bioengineered implants, cell therapies, and transplantation. Furthermore, the intravascular innate immune system can cause severe side effects in patients, as well as rejection and dysfunction of implanted devices and tissues. In this project, our focus has been on end-stage renal disease (ESRD).
Chronic kidney disease is a major cause of ESRD worldwide: 800,000 patients in Europe and in the US, respectively, require long-term treatment, initially with peritoneal dialysis, and then haemodialysis (HD) and kidney transplantation. An ESRD patient on haemodialysis has poor quality of life, with an average life expectancy of only 4 years and healthcare costs of ≈€40,000 to €80,000 per year. Kidney transplantation totally changes life for an ESRD patient, who can then return to normal life, but more widespread adoption of this treatment is still hampered by the low number of available kidney grafts.
Both HD treatment and transplantation are associated with severe adverse reactions that are caused by thromboinflammation. These reactions are triggered by the intravascular innate immune system (the complement, coagulation and contact systems), which is activated by contact between whole blood and the biosurfaces in the kidney (ischemic cells) and throughout the extracorporeal circuit (the extensive biomaterial surfaces).
HD is a life-saving treatment modality in ESRD, but it also represents a major challenge to the intravascular innate immune system, and chronic inflammation is strongly associated with cardiovascular disease (CVD) in HD. The contact between whole blood and biomaterial in the extracorporeal circuit leads to contact activation of proteins within the plasma cascade systems that results in thromboinflammation during dialysis. The contact activation initially leads to a local generation of inflammatory mediators on the material surface. This inflammation is further spread by soluble activation products and mediators (e.g the anaphylatoxins C3a and C5a, bradykinin, and various cytokines and chemokines) generated during dialysis and transported in the extracorporeal circuit back into the patient, together with activated leukocytes and platelets. The combined effect is activation of the patient’s cardiovascular endothelium, which loses its anti-thrombotic and anti-inflammatory properties leading to atherogenesis and arteriosclerosis. Because of this complex inflammatory response, it is conceivable that maximum suppression of the intravascular innate immune system is needed in order to decrease the risk for CVD in HD. This goal can be achieved either by treating the patients with systemic drugs of broader specificity than the anticoagulants used today, targeting more than one of the intravascular cascade systems, or alternatively by employing “stealth” biomaterials that cause minimal cascade system activation.
To date, kidney transplantation is the most effective treatment for patients with ESRD. Despite impressive improvements in treatment results since its introduction in the 1960s, there is still an average graft loss of 25% after 5 years and almost 50% after 10 years. The main target for improving kidney transplantation results, is a thromboinflammatory reaction known as ischemia/reperfusion injury (IRI), since ischemia followed by reperfusion evokes acute renal failure, delayed graft function, and remote organ failure. Immunosuppression does not affect the humoral innate immune system to a great extent and therefore does not attenuate the IRI. The transplantation field is also hampered by organ shortages. One option for increasing the donor pool is an increased use of marginal organs, such as those from deceased-after-cardiac-death (DCD) donors. However, these organs are also severely affected by IRI.
2. Aim
The overall aim of this project was to clarify the mechanisms of thromboinflammation and to identify its own natural specific control points of regulation in these adverse reactions in order to be able to significantly improve the quality of HD devices and kidney grafts by applying these concepts to the regulation of HD and kidney transplantation.
a.) To identify points of regulation in the cross-talk between blood (cascade systems, blood cells) and material/cell surfaces, in order to identify targets for therapeutic regulation of the intravascular innate immune system.
b.) To reduce innate immune activation by the administration of soluble molecules that inhibit the complement cascade including the major objective to to bring a prototype peptide complement factor C3 inhibitor, AMY-101, to clinical trial as a contributor to the regulation of thromboinflammation.
c.) To silence or minimize the activation of intravascular innate immune reactions by creating nano-geometrical surface profiles and regulators that can be used as coatings in devices and organs. Inhibitors that are easy to apply and applicable to a broad range of materials and cell surfaces have been constructed.
Project Results:
3. Identification of new points of regulation in thromboinflammation (WP2, WP5)
Thromboinflammation is a complex reaction that involves the intravascular innate immune system, which consists of the cascade systems of the blood (coagulation, contact, complement, and fibrinolysis systems) and cellular components such as polymorphonuclear (PMN) cells, monocytes, NK cells and platelets. The regulators of thromboinflammation that are used in the clinic today belong to the anticoagulant family, with inhibitors of thrombin and FXa as the main new members. Newly introduced inhibitors are eculizumab that blocks complement factor C5 and inhibitors of kallikrein, bradykinin receptor 2, and FXIIa. The role of the latter inhibitors has not been fully evaluated in thromboinflammation. We have focused on identifying points of thromboinflammation which involve the cross-talk between the respective cascade systems and the cellular components and investigating the possibility of using nanogeometrically modified materials in order to minimize thromboinflammatory reactions on biomaterials.
3.1a C3(H2O) as a ligand of CD11b in the formation of platelet/leukocyte complexes
The formation of complexes between PMNs and platelets is of substantial importance for the occurrence of thromboinflammation. The generation of these complexes is therefore a potential target in order to regulate thrombinflammation. In PMN-platelet complex formation, the expression of CD11b/CD18 (Mac-1) on PMNs is a prerequisite. In basic science experiments, we have identified two new ligands for CD11b which are important points of intervention in the cross-talk between PMNs and platelets. One is C3 in the form of C3(H2O), and the second is glycoprotein GPIb, both expressed on platelets. We have also further characterized developmental endothelial locus-1 (Del-1), a third ligand for CD11b with inhibitory functions, with regard to thromboinflammatory regulation.
In view of the importance of the aforementioned interactions, we employed whole-blood models and examined the effect of candidate innate immune regulators on the interactions of platelets with PMNs.
3.1a i) Formation of PMN/platelet complexes (PPC) in whole blood
As we previously reported, the formation of PPC can be triggered by thrombin receptor-activated platelets in combination with platelet-mediated complement activation. Consistent with our previous data, activation of whole blood with 33.5 µM thrombin receptor-activating peptide (TRAP)-6 led to PPC formation (CD42a-positive PMNs; Fig 1A). The conjugate formation was triggered by complement activation and was significantly inhibited by the addition of the complement (C3) inhibitor Compstatin and a C5aRA (p<0.05; n=5). Conjugate formation was also inhibited by the anti-C3a monoclonal antibody (mAb) SD17.3 (p<0.0001; n=5) as well as anti-CD11b (p<0.01; n=5) and anti-P-selectin (CD62P) (p<0.0001; n=5) mAbs. Anti-CD11b inhibited conjugate formation by 65-75%, whereas anti-C3a and anti-CD62P completely abolished PPC formation. A mAb against activated Mac-1 (CBRM1/5), which inhibited the binding by 85% (n=3), confirmed the interaction with CD11b/CD18 (Fig 1B).
3.1a ii) Complement proteins bound to platelet microparticles (PMPs)
Platelet microparticles (PMPs) were shown by flow cytometry to expose C1q, C4, and C3. The C3 results were confirmed by Western blotting using the polyclonal anti-C3c antibody. The blots showed intact alpha- and beta-chains and the C-terminal alpha-chain 45-kDa fragment, consistent with the forms of C3(H2O) previously reported on activated platelets.
3.1a iii) Formation of PMP/PMN complexes
PMPs isolated from fresh serum were added to blood-cell preparations with a reduced number of platelets and depleted of plasma proteins, and the binding of CD41a+ PMP to PMNs was analysed by flow cytometry. Upon activation with 5 nM C5a, a high percentage of the PMNs bound to CD41a+ PMPs. The PMNs bound significantly more microparticles after activation than did activated cells without added PMPs (p<0.001; n=6).
As is true for PPC formation, PMP complex formation with PMNs was also inhibited by the anti-CD11b, anti-C3a, and anti-CD62P mAbs. Anti-CD11b (p<0.01; n=9) and anti-C3a (p<0.05; n=5) mAbs significantly reduced the formation of PMP-PMN conjugates (to background levels) when compared to non-activated cells that had been incubated with PMPs. Anti-CD62P mAb lowered the PMP-PMN complex formation even further, to levels comparable to those obtained when no PMPs were added (p<0.0001; n=9). PMP-PMN complexes were unaffected by the addition of control Ig.
3.1a iv) Interaction of various forms of C3(H2O) and CD11b/CD18
The interaction between C3 and CD11b was studied with the quartz crystal microbalance with dissipation (QCM-D) technique using purified native C3 and rMac-1 (CD11b/CD18). This is a technique in which one ligand (of a putative ligand-receptor pair) is adsorbed to a sensor surface, while the other ligand is allowed to flow over the surface, enabling binding.
Native C3 was adsorbed to a hydrophobic polystyrene-coated QCM sensor surface, after which the binding of recombinant Mac-1 (rMac-1) was monitored. IgG and casein served as negative controls. The mass of bound rMac-1 to the different surfaces clearly demonstrated a binding of rMac-1 to the C3-coated surface, whereas no binding or only trace amounts of rMac-1 bound to casein and IgG, respectively. The incubation of rMac-1 with increasing concentrations of soluble iC3b prior to addition to the C3-coated surface inhibited the binding of rMac-1 at 1:1 ratio by 20%; at 5:1 by 45%, and at 10:1 by 60%. Similar experiments employing anti-CD11b mAbs showed inhibition of binding by 70-80%.
3.1b Functional testing of Del-1 in thromboinflammation
Developmental endothelial locus-1 (Del-1) was identified a few years ago as an inhibitor of leukocyte integrins, including CD11b. Given the role of CD11b in platelet-leukocyte interactions, we examined the role of the endogenous anti-inflammatory molecule Del-1 on the shaping of platelet-monocyte interactions. To this end, we assessed the effect of Del-1 on the modulation of platelet-leukocyte aggregate formation. Specifically, we employed a human whole blood model and incubated isolated islets of Langerhans. This model represents a whole-blood model of the immediate blood-mediated inflammatory reaction (IBMIR). The exposure of blood to isolated islets of Langerhans induced the activation of coagulation, as measured by the generation of thrombin-antithrombin (TAT) complexes. Moreover, the exposure of blood to islets resulted in increased formation of platelet-monocyte aggregates, a major indicator for the activation of thromboinflammation. Interestingly, the pre-treatment of blood with Del-1, prior to the addition of islets, led to decreased levels of platelet-monocyte aggregates, as well as inhibition of the coagulatory response (Fig. 2).
This heterotypic platelet-monocyte interaction was dependent on the binding of the platelet glycoprotein GPIb to the leukocyte integrin Mac-1. Moreover, we found that the N-terminal EGF-like domains of Del-1 were sufficient to block the GPIb-Mac-1 interaction.
Importantly, the presence of Del-1 was also associated with lower levels of C-peptide, thus corresponding to better islet integrity and function. Therefore, the presence of Del-1 in this whole-blood model of IBMIR substantially decreased the IBMIR-associated thromboinflammatory response. These findings clearly suggest the value of a targeted use of Del-1 for the elimination of the intravascular thrombo-inflammatory effects that are elicited upon the exposure of the host to foreign (bio)surfaces, as well as in the context of inflammatory responses to transplantation.
3.2 Activation of the lectin pathway
The lectin pathway (LP) of complement has been linked to the coagulation system in several studies, and inhibition of mannan-associated serine protease (MASP)-2 in vivo affects the coagulation system. Two proteases in the LP, MASP-1 and MASP-2, both cleave coagulation factors. MASP-1 has thrombin-like properties, while MASP-2 cleaves prothrombin to active thrombin. All these experiments have been performed in purified or semi-purified systems and no mechanistic explanation has been presented for how the LP is activated under these circumstances.
The objective of this study was to clarify the not-yet identified mechanisms involved in triggering the activation of the LP during thrombotic reactions. Novel sandwich ELISAs for the detection of complexes between MASP-1 or MASP-2 and the serine protease inhibitors (serpins) C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. Activated platelets were shown by flow cytometry to bind the recognition molecules ficolin-1, -2, and -3 (but not mannan-binding lectin [MBL]), and the binding was associated with an activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma both bind and activate MASP-1 and MASP-2. As demonstrated by ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus (SLE) patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. In conclusion, MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions both in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation. (Fig 3).
3.3 Nanostructured materials
Based on preliminary data, we hypothesized that thromboinflammation induced by biomaterials would be affected by not only the chemical nature of the material per se but also by the nanogeometry of the surface involved. Various types of nanoprofiled material surfaces (including those produced using molecular imprinting [MIP]) were produced for clarification of the fundamental conditions for complement and contact system activation on material surfaces with respect to nanogeometry as well as for potential clinical applications involving materials in contact with blood.
3.3a Production of nanostructured materials
Various nanostructured materials have been designed, fabricated, and characterized. Sparse colloidal lithography and sputter deposition were combined to produce samples on glass and silicon wafer substrates. The nanotopography was varied in terms of particle type (silica and polystyrene [PS]), particle size (34-60 nm in diameter) and inter-particle distance. Furthermore, in addition to the topographical features, all the produced materials allowed for defining various surface chemistries. The particles were analysed using nanoparticle (NP) tracking analysis prior to deposition. Optical readout for plasma protein binding was performed on glass samples using UV-visible spectrophotometry. Sets of samples were available for partners in WP2 and delivered to UU). The sets included the following sample surfaces: S1, Flat control; S2, 34 nm SiO2 NP deposited from MilliQ water; S3, 60 nm PS NP deposited from MilliQ water; S4, 60 nm PS NP deposited from 3mM NaCl, wet baked at 110°C; and S5, 60 nm PS NP deposited from NaCl, dry baked at 110°C. All sample surfaces (S1-S5) were covered with Ti and Au. The surfaces were then thiolated to obtain self-assembled monolayers (SAMs), which ensured identical surface chemistry on all sample surfaces, independent of their nanotopography. For the first set of experiments, HS-C11-OH was used. Other types of SAMs were also planned to be used but remain to be tested.
3.3b Generation of nano-geometrical profile material surfaces by applying MIP
Efforts have focused on the design, synthesis, and physical characterization of a series of polymeric nanostructures for subsequent evaluation of their impact on the complement and coagulation cascades. The primary variables being examined with respect to complement activation are morphology (with thin film-based nanowires, nano-fibers, and, as reference, various films devoid of hierarchical architectures are being studied) and the influence of inherent polymer physico-chemical properties. Our work in another project aimed at the development of protein-resistant surfaces (Norwegian Research Council, NFR) identified sulfobetaine ([2-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide) as a candidate for initial morphologic studies within DIREKT, on the basis of its “mixed-mode” characteristics (somewhat hydrophobic and zwitterionic). In parallel, work that has been accepted for publication in Biomaterials is underway to identify other polymer systems with different physico-chemical properties for the development of a series of second-generation materials. An initial technical challenge was the up-scaling of the material synthesis from QCM chip surfaces (still used here for screening for protein binding) to the glass slide format. After feedback from DIREKT partners based upon the assay results, the procedure was modified, this time including an additional reference system (prepared in a newly developed non-ionic eutectic mixture [melt]) and additional examples of each system (n=4). Also, studies based upon surfaces molecularly imprinted with heparin have been completed and published in Biomaterials Science.
3.3c Definition of the functional requirement for the recognition and activation of the complement and contact systems
A series of experiments has been done to evaluate the conditions for the recognition and activation of the complement and coagulation activation systems by the above-prepared nanostructured materials (3.3a and 3.3b) in contact with whole blood. These experiments were done by mounting the nanostructured materials onto a 2-well slide chamber model filled with fresh blood from healthy donors which was incubated with rotation for 60 min at 37°C. In one of the wells, 0.25 IU/mL heparin was added, and 0.5 IU/mL heparin was added to the other. The platelet count was measured after incubation, and no or very little loss of platelets occurred for all the materials. Plasma from all the samples was then analysed by ELISA to assess coagulation and complement activation products. All experiments were repeated at least three times.
There were clear differences in protein adsorption and blood activation between the different nanostructures generated in 3.3b (Fig 4).
For these measures, surfaces were synthesized in 3.3a with identical chemistry but different nanoprofiles. The material surfaces coated with closely packed 60-nm particles, as well as flat control surfaces, were found to be highly thrombogenic when compared to surfaces coated with sparsely packed particles of the same size or smaller. A reciprocal relationship was found regarding the initial inflammatory response; i.e. the most thrombogenic surfaces were the lowest complement activators (Fig 5).
Furthermore, huge differences in C1q binding from human EDTA-plasma were demonstrated. The surface with the smallest structure (S2, 34 nm) bound minimal amounts of C1q, i.e. like the negative control (PVC). All surfaces coated with larger NP (60 nm) bound C1q as well as did the positive control (a heparin-coated polystyrene surface), irrespective of the packing density of the NP. Coagulation Factor XII was close to or below the detection limit on all NP-bearing surfaces (S2 - S5), as well as on the negative control. The flat surface (S1) bound significant amounts of FXII, at approximately half the levels of the positive control (Fig 5).
In order to further address the question of protein adsorption on curved surfaces, we developed a novel technique for determining NP interactions with various purified proteins in solution. Particles of different sizes were first coated with a protein antigen and then with antigen-specific IgG. Finally, purified C1q was added, and the bound amount was quantified. Higher amounts of both IgG and C1q were detected on the smaller NP with a size in the same order of magnitude as the proteins, as compared to the larger particles, which have a flatter structure.
In addition, we have initiated plasma protein profiling to assess protein corona adsorbed to the various nanostructures in order to define the requirements for recognition and activation of the complement and contact systems at the protein level.
3.3d Characterization of biotin-avidin binding surfaces by applying MIP onto material surfaces
We have published a strategy for the development of biotin-avidin binding surfaces in an ‘ultra-thin film format’ (3-5 nm) in the Journal of Nanobiotechnology. The composition of the polymers was derived from earlier work using a combination of molecular dynamics-based studies of potential pre-polymerization mixtures and radioligand studies of the corresponding bulk polymers. Evaluation of ligand-biotin selective polymer film recognition properties was undertaken using QCM, and appropriate control systems were employed.
The biotinyl moiety-selective molecularly imprinted polymer-grafted SAM-Au/quartz nano-films showed significant selectivity for biotinyl-bearing carbohydrate and peptidic structures and could efficiently distinguish the biotinylated structures from the native counterparts (Fig 6, upper panel). The interaction of the analyte with imprinted sites through the formation of affinity complexes was determined by calculating the apparent stability constant (Ks=ka/kd) and showed a two-fold higher stability for binding to the MIP than to the reference (Fig 6, lower panel).
Because of the frequent problems we encountered with the mechanical stability of the polymer films, we devoted our efforts to developing the use of silanized surfaces and organic modifiers (low concentrations of organic solvents) to enhance the yield from the fabrication process. A series of second generation of biotinyl moiety imprinted materials have been synthesized in two formats, ultra-thin film and with hierarchical architectures derived from sacrificial meso-structure (mono-disperse polystyrene). The modified surfaces have been characterized by reflection absorption infrared spectroscopy (RAIRS), scanning electron microscopy (SEM), atomic force microscopy (AFM), ellipsometry, and X-ray photoelectron spectroscopy (XPS).
4. Development of regulators of thromboinflammation to be used in hemodialysis and kidney transplantation (WP3, 4, 5)
One approach to regulating thromboinflammatory reactions on biosurfaces in contact with whole blood (such as occur during haemodialysis on the biomaterial surfaces and in transplantation on cell surfaces during IRI) is to precoat the surface with a non-reactive shield or a regulator of the individual cascade systems or cells. We have investigated a number of alternative coatings that use different mechanisms of regulation and have identified some of them as candidates for further development into clinically applicable treatment modalities. For instance, we have developed various types of PEG and heparin derivatives for both direct protection of biosurfaces and also to be used as linkers of protective peptides and macromolecules to biosurfaces.
4.1. PEG coatings
4.1a. Coating of cell surfaces with a PEG-lipid construct
Evaluation of different PEG constructs on various cell surfaces was performed. Human mesenchymal stromal cells (MSCs) were coated with a PEG-phospholipid construct and showed less platelet consumption and a decrease in activation of the coagulation and complement systems when tested in an in vitro whole blood model. These findings were confirmed after coating of hepatocytes with PEG (Fig 7). This promising inhibitor of thromboinflammation was further tested in small and large animal models and the intension is to bring this technique to the clinical transplantation. See also 8.2.
4.1b. Development of material-coating techniques with PEG and heparin
In addition, we developed material-coating techniques with PEG. For instance, Pluronic F108 was adsorbed to hydrophobic surfaces (such as PS and PVC), resulting in diminished activation of the coagulation and complement systems. Efforts were also focused on the development of a thicker coating that could better shield the surface than did Pluronic F108, thereby more thoroughly decreasing the biomaterial-induced adverse effects. To this end, Pluronic molecules were polymerized using multi-armed PEG molecules covalently attached to the Pluronic. This conjugation approach resulted in further decreased activation of both the coagulation and complement pathways.
Also, a heparin-conjugate and unfractionated heparin were attached through heparin-binding peptides (HBPs) conjugated to Pluronic F108 that was adsorbed to a material surface. This approach resulted in diminished activation of the coagulation and complement systems. Alternatively, we used four-arm PEG-maleimide, to which HBPs was attached (Fig 7). Heparin-conjugate or unfractionated heparin (UFH) was then captured on the surface by peptides. Also, a heparin conjugate was designed by covalently attaching unfractionated heparin to multi-arm PEG, which was immobilized on the surface that was pre-coated with Pluronic-HBP. This composite was achieved either by first attaching the eight-arm PEG to the Pluronic and then adding UFH or by first making the eight-arm PEG+UFH conjugate and attaching it to the surface with Pluronic-HBP. Both approaches were efficient, as shown by heparin quantification.
4.2. Factor H-like inhibition
Factor H (FH) within the alternative pathway (AP), is one of the most powerful natural inhibitors of complement activation. Within DIREKT, two strategies have been developed to engage this inhibitor. The first principle was to use a peptide with affinity for FH to pull down the inhibitor to the biosurface. The second was to truncate the FH gene so that only the active sites in the N- and C-terminal parts of FH were expressed, with the resulting molecule being designated “mini-FH”. We have achieved significant progress in the development of the peptide 5C6 and the complement inhibitor mini-FH, which are among our main deliverables.
4.2a. Evaluation of the 5C6 molecule
The 5C6 molecule, a peptide with the ability to bind and recruit FH to sites of complement activation, was developed and tested in a mouse model of arthritis.
The peptide 5C6 showed high selectivity for FH and binds FH from different species, i.e. human, monkey, pig, rat, and mouse. This finding was particularly important in the context of the DIREKT project, given the inclusion of mouse and pig models in various projects. Further, we have established a surface plasmon resonance (SPR)-based assay and quantified the monovalent binding affinity of 5C6 to FH with a KD of ~150 nM. This assay was also used for the screening of an alanine scanning library of 5C6, revealing that several amino acids in the cyclic part and at the C-terminus, but not in the N-terminus of the peptide, drive the interaction with FH. Using site-specific truncation and de-novo synthesis, we showed that truncated analogs lacking the N-terminus have comparable activities as the full-length peptide. Because of its shorter size and more effective synthesis, we therefore selected truncated 5C6 as our new lead compound. Furthermore, using the SPR assay, we narrowed down the binding region of 5C6 to FH8-15. Apart from the molecular and in vitro studies, we conjugated 5C6 with a peptide able to bind the synovial membrane and used this construct in a mouse model of antigen-induced arthritis. Our data showed that the dual peptide was able to bind to inflamed synovium in vivo and recruit FH to the inflammation sites, inhibiting swelling and migration of inflammatory cells in the arthritic mice.
4.2b. Mini-FH inhibitor
Regarding the mini-FH inhibitor, we showed that it maintains or exceeds the host-cell protective activity of FH despite its 70% reduction in size. Human mini-FH showed inhibitory activity in pig plasma in collaboration with WP8, supporting the suitability of mini-FH for in vivo studies. Furthermore, our pharmacokinetic studies performed both in mice and non-human primates revealed that although a rapid initial clearance of the inhibitor after intravenous injection, residual protein could be detected much later, indicating longer cell/tissue residence when compared to the circulatory half-life. Several collaborative animal experiments have been initiated for human mini-FH, e.g. with WP5. A structural comparison of several regulator homologs bound to C3b has been published, revealing several determinants that define complement-inhibitory activity and adding useful insights for the design of next-generation inhibitors. In addition, other FH variants, i.e.; FHL-1, FHΔ10-15, and midi-FH, were engineered, expressed, and tested to evaluate their ability to inhibit the deposition of C3b triggered by the lipopolysaccharide (LPS)- or paroxysmal nocturnal hemoglobinuria (PNH) cell-induced AP. This comparison indicated that FHL-1 is responsible for about one-third of the systemic complement regulation of the AP, a finding that has implications for the pathophysiology of diseases associated with genetic variations within the FH protein family. It also showed that the binding affinity for C3b is correlated with the number of potential binding sites, and that FHΔ10–15 and midi-FH are substantially more active AP regulators than is mini-FH, in assays employing LPS but not PNH cells, suggesting that the mechanism of action of these proteins is subject to different limitations when they are acting on host/self versus foreign (or artificial) surfaces (Fig 8).
4.3. C1s inhibitor
Apart from AP inhibitors, we also recombinantly expressed the classical pathway (CP) inhibitor BD001 in yeast (P. pastoris) and revealed strong binding affinity for C1s and CP-inhibitory activity in human plasma. Unfortunately, BD001 development was hampered by the heterogeneity of the yeast-derived product. We then attempted to express the protein in expression systems that facilitate folding (BD001 has 20 cysteines) and allow for proper post-translational modification (sulfation contributes to its overall activity). The use of mammalian cells (HEK293T) resulted in expression but no secretion of the protein. A baculovirus system was also evaluated with two different cells (SF9, Hi5), but the desired protein was not expressed. In the meantime, a group from Australia was able to express BD001 (Gigastasin) in High Five insect cells in large enough quantities to resolve the crystal structure of the inhibitor in complex with C1s. Gigastasin appears to block the active sites of C1s and MASP-2 and was able to inhibit the activation of the CP and the LP pathways, supporting the suitability of BD001 for use as a complement inhibitor.
4.4. C4BP-peptides
We also characterized several peptides, i.e. P1, CKENQGKLEKLELDYLKKL; P2, CDKAMKAKVATYLGGLPDVPFSAT; P3, CSGIAQFHIDYNNVSSAEGWHVN; and P4, CGVQLDLDEAI in terms of their ability to recruit C4BP and inhibit CP activation. These peptides were all conjugated to maleimide-PEG-lipid and used to coat cell surfaces. P1, P2, and P3 were considered potential therapeutic candidates, since they showed binding to C4BP by QCM-D; in contrast, only minor C4BP recruitment was achieved using P4.
5. Development of assays for assessment of mouse and porcine complement (WP5 and 6)
The cascade systems of the blood have traditionally been investigated in humans and in human blood. Assays to assess the activation of the systems in other species have been difficult to obtain. DIREKT partner Hycult Biotech has developed assays for complement analyses in mouse and pig, the most common mammals for in vivo research.
Development of immunoassays to detect activation of the mouse complement system was a main objective of WP5. Partner Hycult Biotech developed four different assays for the assessment of specific activation products and pathways of complement system, including detection of activation of the CP, AP, and LP. All assays successively passed quality control and testing within the consortium.
The deliverable was to establish three functional complement pathway assays (for the CP, AP and LP) and an assay for the porcine activation fragment C5a. All four assays were successfully established and met all the requirements for assay performance and quality control, as determined according to internal standards. Results from independent validation experiments showed that assay performance is good and that the achieved data are accurate and reliable. The CP and LP assays are now commercially available; the AP and the C5a assays are expected to become available early in 2018 (Fig 9).
6. Testing of inhibitors of thromboinflammation in vitro and in small animals (WP5)
In order to further evaluate promising regulators of thromboinflammation that are candidates for further development and in vivo large animal testing, the treatment modalities were tested in vitro and in vivo in small animal models.
6.1. Inhibition of thromboinflammation in IBMIR
We tested the developed inhibitors of intravascular innate immunity using in vitro and small animal models. The effect of innate immune regulators on the modulation of thrombo-inflammatory reactions was studied by establishing whole-blood models. We focused on the instant blood-mediated inflammatory reaction (IBMIR), which is elicited upon islet transplantation (Tx) and causes islet dysfunction. We used a model in which human whole blood is incubated with isolated porcine islets of Langerhans and a model in which whole blood is in contact with porcine endothelium; both models elicit an IBMIR-like inflammatory reaction, including a strong thrombo-inflammatory response. In this context, we studied the role of endogenous anti-inflammatory agents or highly specific complement inhibitors as possible therapeutic interventions for thrombo-inflammatory reactions (including leukocyte adhesion, leukocyte-platelet interactions, and activation of coagulation). For instance, Del-1 was tested in these models and was found to be a promising inhibitor of thromboinflammation. Using these models, we focused our activities on the immobilization of regulators on cellular surfaces and (bio)surfaces, including regulators of the complement system such as apyrase, a regulator of platelet and coagulation activation, and heparin-binding peptides. Importantly, co-immobilization of regulators on the surface of different target cells led to attenuated thrombo-inflammatory responses upon exposure of these cells to human blood.
6.2. Coating endothelial cells with heparin
A conjugate of multiple unfractionated heparin chains coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC (a conjugate of 70 heparin molecules linked to a polymer chain) to protect wild-type (WT) and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNF-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation, and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNF-treated PAEC with CHC (100µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged the clotting time of human blood (115.0±16.1min p<0.001) when compared to WT PAEC (34.0±8.2min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3±11.3min p<0.001; GTKO.hCD46.hTBM: 146.2±20.4min p<0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.
To further study the effect of the heparin coating on the regulation of the adverse effects caused by exposure of the host to (bio)surfaces, we established a mouse Tx model of ischemia-reperfusion injury and a mouse model of islet Tx. We studied the function of heparin coating and of complement regulators. Of note, CHC improved the function of the graft in mice that received a kidney transplant after cold storage; this effect was accompanied by inhibition of inflammation. Together, these data suggest that single or combined use of innate immune regulators coated on biosurfaces may be advantageous for the elimination of the thrombo-inflammatory effects elicited upon exposure of the host to foreign (bio)surfaces.
7. Testing of inhibitors of thromboinflammation in large animal models (WP6)
Pig models of kidney transplantation were used to evaluate selected promising inhibitors of thromboinflammation, with the aim of abrogating the host response to the transplanted organ with regard to intravascular innate immunity and IRI.
7.1 In vitro and ex vivo models of kidney IRI and antibody-mediated rejection
Our specific objective here was to establish ex vivo models to study pathophysiology related to kidney transplantation and to apply regulators to dampen the host response. Two complementary models were established, one based on cultured endothelial cells (EC) in vitro, and another on isolated ex vivo porcine kidneys. The aim was to characterize and apply regulators to these model systems under conditions of IR and antibody-mediated rejection (AMR). In the cell-based assay, hypoxia and subsequent reperfusion with fresh human whole blood mimicked IRI; inclusion of antibodies against EC simulated AMR. IR in isolated porcine kidneys was accomplished by overnight cold storage of the explanted organ and subsequent reperfusion with autologous blood. In separate experiments, fresh human whole blood was employed to trigger a xenoreaction simulating AMR. Complement activation was a major readout in the EC model; a broad panel of thromboinflammatory read-outs and markers of kidney injury and function were characterized in the isolated organ model. Regulators, primarily PEG-lipid and mini-FH, were administered in the models. The main findings were that PEG-lipid lowered platelet consumption in AMR of isolated kidneys. The sampled biological material from the mini-FH study are currently under analysis.
8. Three main potential clinical treatment modalities resulting from DIREKT project activities
8.1 Development of the C3 inhibitor AMY-101 from prototype to in-human drug
The initial focus of Amyndas in the DIREKT was to bring AMY-101 (of the Compstatin family) from a preclinical drug candidate to the stage where it can be tested in a clinical trial with the indication of ABO-incompatible kidney transplantation.
Our initial focus in the DIREKT project was on the design and conduct of the appropriate pre-clinical studies to evaluate the safety of the C3 complement inhibitor AMY-101 and its pharmacokinetic (PK) profile before its application in human clinical trials. Therefore, we designed our preclinical strategy and proceeded to the manufacturing of preclinical grade AMY-101 and the conduct of GLP toxicology studies. The preclinical toxicology studies in non-human primates showed that AMY-101 did not show any systemic toxicity after repeated administration, and the GLP PK study showed a good PK profile, which can be used to guide the human clinical studies. In the meantime, GMP grade (clinical grade) AMY-101 was produced, to be used in the human clinical studies and long-term stability studies were initiated to evaluate the stability over time.
In Q1 2017 Amyndas submitted a clinical trial application for the first in human (phase I) clinical study to the US FDA. The FDA approved the application while in the meantime, the stability studies confirmed long-term stability of AMY-101. Following FDA approval, Amyndas started the phase I clinical trial in human volunteers and successfully completed the study in November 2017.
This study involved testing AMY-101 in human volunteers, to establish the safety and PK/PD profile of the drug. The study comprised of a single ascending dose (SAD) part and a multiple dose (MD) part. In the SAD part of the study single doses of AMY-101 were administered systemically through the subcutaneous and intravenous routes, and followed for 21 days of monitoring. In the MD part of the study multiple doses of AMY-101 were administered by subcutaneous administration, and the subjects were followed for 14 days after last dosing. AMY-101 was well tolerated in both the SAD and MD part of the study. No serious adverse events of AMY-101 have been observed in any of the subjects dosed. Analysis of the PK/PD profile showed that saturation of target C3 is reached after one administration at the pharmacologically active dose, and this saturation can be maintained with daily dosing at this level.
At the same time Amyndas prepared the phase 2a clinical study protocol,l for the application of AMY-101 in ABO incompatible kidney transplanted patients.
In summary, during the duration of the DIREKT project, all the objectives and milestones of WP8 have been successfully completed.
8.2 Demonstration of the potential clinical benefit from PEG-lipid coating with regard to thromboinflammation (IRI) in a porcine in vivo allogenic transplantation model
The objective was to establish a porcine in vivo survival model of kidney transplantation, to characterize the model, and to apply a regulator to evaluate its effect on kidney function and survival (task and deliverable). The model, which was established at month 12 (a milestone), is based on explantation of kidneys from a donor pig, cold preservation of the explanted kidneys, and subsequent transplantation into a swine leucocyte antigen (SLA )-mismatched recipient, both of whose native kidneys had been removed. The recipients were under continuous surveillance, were clinically examined regularly, and blood, urine, and tissue samples were regularly obtained. Plasma, urine, and tissue were examined for a broad panel of thromboinflammatory read-outs, and markers of kidney injury and function were measured. The PEG-lipid was extensively evaluated in this model, and the regulator was then administered to the explanted kidneys closely immediately before transplantation. Donors who received PEG-lipid-treated kidneys had lower levels of complement and coagulation activation, as compared to vehicle-treated controls. Pro-inflammatory cytokines (IL-1beta, IL-6) were also lower, and kidney function was improved. The intension is now to bring this technique to the clinic.
8.3 Development of a haemodialysis catheter, applying novel knowledge concerning the effect of nanogeometry on thrombinflammation
As a clinical application of the studies of the effect of the nanogeometry on thromboinflammation, we successfully developed fabrication approaches to coat multiple catheter materials with nanostructures and tested the functionality of the coatings to reduce blood activation and bacterial adhesion. Nanostructuring approaches were developed to prepare pure nanotopographic profiles (30 to 140-nm profiles were prepared) or multifunctional coatings containing nanotopographic profiles, noble metal chemistries, and anti-coagulants. These coatings were assessed on catheter materials and on commercial catheters. These prototype coatings on commercial catheter materials were tested for blood compatibility (using the Chandler loop test) and bacterial adhesion (using the Ahearn test). Several multifunctional coatings showed significant reductions in blood activation (TAT level, blood platelet count, C3a level, and sC5b-9 level) and bacterial adhesion when compared to the same commercial catheters in uncoated form.
A coating was developed which significantly reduced both blood activation (by >95%) and bacterial adhesion (by >90%) when compared to the same commercial catheter, uncoated. This coating is now intended to be CE-marked and commercialised for haemodialysis catheters.
9. Exploitation and dissemination (WP10 and WP11)
Other objectives of DIREKT were the transferral of information and the identification of a series of questions related to ESRD with regard to academic and industrial issues. A number of activities were carried out to achieve these goals.
9.1. Aegean Conferences on Biosurfaces (WP10)
The partners of the Aegean Conferences have developed expertise during the past 18 years in the organization of scientific conferences. Over the years, these conferences have fostered dissemination, training, and scientific dialogue in disciplines related to the central theme of the DIREKT consortium.
In accordance with the Exploitation and Dissemination plan of the DIREKT consortium, Aegean Conferences sponsored two conferences under the titles ‘1st and 2nd International Conferences on Immune Responses to Biosurfaces: Mechanisms and Therapeutic Interventions’. The first conference took place from September 27 to October 2, 2014 in Chania, Greece, and the second from September 26 to October 1, 2016 in Rhodes, Greece (Fig 10). In both of these face-to-face meetings, explorations of innovations and inventions were exchanged among all of the WP leaders (and other representatives of the consortium) and leaders of industries dealing with ESRD-related treatment modalities. The conference programs can be found on first conference and second conference. Collectively, these meetings were attended by over 100 participants, including the DIREKT partners from academia and industry as well as the members of the Advisory Board of DIREKT, who presented their progress. In addition, WP-focused discussions took place during and after the meetings. As is typical of the style of the Aegean Conferences, there were several awards conferred on young trainees who presented their work during these conferences.
9.2 Publication of conference proceedings
Directly relevant to the objectives of WP10 is the publication of the proceedings of the first conference that appeared in Advances in Experimental Medicine and Biology (vol. 865, 2015).
9.3 Dissemination and exploitation (WP11)
The DIREKT project has been very productive in terms of dissemination and exploitation activities, generating 126 per-reviewed publications, one academic PhD thesis, 130 dissemination activities (such as oral presentations and posters at international conferences and scientific meetings), and 8 exploitable foregrounds during the 4 years of the consortium. It has also established a large number of fruitful collaborations among the partners involved, in both academia and industry, that will be continued after the project period ends.
10. Conclusion
We have clarified several mechanisms and identified specific control points of regulation in adverse reactions caused by thromboinflammation and in order to be able to significantly improve the quality of haemodialysis devices and kidney grafts we have developed new concepts of regulation in haemodialysis and kidney transplantation. The results of this research and development have been applied in three distinctive clinical treatment modalities and in assays for analyses of thromboinflammation in animal models:
1. We have brought a novel soluble complement inhibitor from prototype drug to a completed phase 1 clinical trial.
2. We have created nano-profiled surfaces with low activating properties that have been applied to catheters intended for haemodialysis use.
3. We have developed a PEG-lipid construct which can be used to coat biomaterial surfaces and have used it ex-vivo to coat kidney grafts intravascularly before transplantation, significantly protecting the kidney grafts against ischemia/reperfusion injury.
4. Finally, we have developed eight assays for the assessment of thromboinflammation in the mouse and pig for use in models of human disease and conditions in which thromboinflammation contributes to the pathophysiology.
All three treatment modalities are at varying stages of development into clinical treatment regimens of ESRD. When applied we anticipate that these modalities will extend the feasible hemodialysis treatment periods and in kidney transplantation attenuate innate immune reactions thereby prolonging the life expectancy of the graft and make more kidneys accessible for transplantation. The impact of this is improved quality of life and lowered costs for treatment of ESRD patients. All the novel techniques can be applied to other types of implantations, extracorporeal treatments and organ transplantations and in the future potentially be used in xenotransplantation and stem cell therapies.
Potential Impact:
11. Impact
11.1 New treatment strategies and improved health
The innate immune system of the blood and other tissue fluids represents a serious obstacle to the development of treatment modalities using tissue transplants and device implants, since it provoke thromboinflammatory reactions. The focus of this project has been the chronic kidney disease known as end-stage renal disease (ESRD).
a.) In hemodialysis patients, the frequent treatments trigger side effects of whole-body thromboinflammation, leading to a dramatically increased risk for cardiovascular disease and for infections. Regulation of the complement, contact and coagulation systems activation, and other aspects of innate immunity will reduce adverse effects in these patients and lower the risk of arteriosclerosis, thrombosis and in severe infections. Today, in their attempts to control the intravascular innate immune system, manufacturers of biomedical device implants are almost entirely restricted to an empirical selection of compounds for biomedical device design.
Development in DIREKT of active easy-to-apply coatings and optimal nanostructured geometries to control immune activation will therefore be of great significance and make it possible to create biomedical devices built of materials that meet the necessary requirements for stability, abrasion, and strength, independent of their inherited properties to trigger thromboinflammation.
b.) Successful transplantation has an immense impact on the quality of life and normalizes the life and health of the patients. The intravascular innate immune system causing thromboinflammation, is involved in various types of rejection during, immediately after, and in the long term after kidney transplantation. This rejection has severe consequences, such as limiting the selection of grafts for specific recipients and reducing the survival rate of the transplanted grafts. The results of DIREKT using soluble complement inhibitors and cell coatings will improve the quality of life for patients with ESRD by optimizing the conditions for kidney transplantation, with the aim of increasing the number of patients that can regain a normal life. In the future, techniques developed within this program may also prove applicable to kidney xenotransplantation (and stem cell therapies), representing a large step forward toward a greater availability of kidney grafts and transplant-related restoration of normal life for ESRD patients.
c.) Also, patients with other diseases such as cardiovascular disease (those receiving stents, cardiopulmonary bypass, heart transplantation) and liver disease (liver dialysis and transplantation), in which transplantation and extracorporeal treatment are important treatment modalities, will benefit greatly from the project.
11.2 A new soluble complement inhibitor (AMY-101)
The phase 1 clinical trial of AMY-101 performed by partner Amyndas is a great achievement and step forward which will greatly promote the development of this compound as an immunoregulatory drug for use in kidney transplantation. AMY-101 has high potential in that it would be a novel inhibitor to target C3 in the complement cascade. Eculizimab, the only clinically available complement inhibitor which targets C5, acts further downstream than AMY-101. Therefore, AMY-101 has a broader effect, inhibiting not only C5a and the terminal complex C5b-9 but also effects mediated by C3 fragments.
The ABO incompatibility barrier is a severe obstacle to transplantation, particularly for living donor transplantations. Recent studies reveal that complement is instrumental in preventing ABO incompatible organ rejection during transplantation. ABO-incompatible kidney transplantation is a possible indication for AMY-101, since it is expected that AMY-101 can make non-matched donor transplantations safer and can lead to accommodation of the graft. This would be of great benefit for patients with ESRD as it could lead to an increased accessibility of donor organs and it would facilitate an increase of the number of transplantations for patients in need.
11.3 Coatings for protection of cells against thromboinflammation
The newly developed coatings of cell surfaces have been shown to significantly protect cells against attack from the innate immune system. In transplantation, the condition primary-non-function is associated with poor long-term survival of the kidney graft and is triggered by the thromboinflammatory reaction called ischemia/reperfusion injury (IRI). The developed coatings lower IRI and are therefore expected to improve the life span of the graft thereby improving quality of life for the patient, increase the number of available graft and lower the cost of the ESRD treatment.
11.4 New bioactive nano- and microstructured materials
The area of nano- and microstructured materials has become a prioritized research direction in Europe, through integrated efforts in FP5, 6 and 7 and in the Nanomedicine European Technology Platform. These efforts have developed knowledge, tools, and frameworks for understanding and design of nanprofiled structures. In DIREKT, our close partnership and integrated approach provides a strong basis for innovation. We have developed robust fabrication routes that can be scaled up to industrial-level processes, enabling the testing of nanostructured materials in medical devices.
a.) One example of this is the new coating developed by the DIREKT partner Bactiguard in collaboration with Århus and Uppsala Universities. This coating combines Bactiguards previous coatings with low thrombinflammatory properties. This will lower the thromboinflammatory reactions in vivo. The surface treatment can potentially also be used in other extracorporeal applications when whole blood comes in contact with biomaterials e.g. heart pumps, cardiovascular bypass etc. The clinical application of this coating is in the near future considering that CE marking is already obtained.
b.) The newly developed protocol based on the findings in DIREKT that will apply nanostructured surfaces in the functionalization of polymer surfaces with artificial avidin, could constitute a potential breakthrough in the field of biomaterial compatibility and would offer a very versatile procedure for adapting surfaces for specific biological challenges.
11.5 New assays for assessment of thrombinflammatory parameters in animal models
Mouse and pig are among the most common and versatile animals for studies of human disease. Partner Hycult has developed four assays for each species. These new assays will replace the previous time consuming and laborious tests, thereby making studies focusing on thromboinflammatory reactions in vivo, more accessible.
11.6 Reinforcement of European SMEs
The four companies that have been involved in DIREKT, has worked closely together with the academic scientists on this project in order to help patients with ESRD to achieve an improved life quality through improved hemodialysis and kidney transplantation. An EU project like DIREKT has also enable the development of personal relations with other participating companies and universities, an invaluable advantage for start-up companies seeking to secure future business relations.
a.) To that end, Amyndas Pharmaceutical, a Greek biotechnology company spun out from the University of Pennsylvania was founded in order to transfer technology from the US to Europe, specifically to Greece. The company develops therapeutics for inflammatory diseases and disorders based on patented technology for modulating the complement system. This company has licensed technology from the University of Pennsylvania and translated it into the clinically applicable AMY-101 for threatment of among other indications, patients with ESRD. This project has been a crucial step for Amyndas in developing a commercial platform for drugs against complement-mediated diseases.
b.) Hycult Biotech is a Dutch SME specialized in the development, manufacturing and commercialization of research reagents (antibodies, immunoassays) in the field of innate immunity. They develop products related to complement, antimicrobial peptides, acute phase proteins, TLR, LPS and microbial toxins. Hycult Biotech further has a specific interest in inflammation and cell - and tissue damage. The DIREKT project has provided a unique opportunity to extend the company’s product portfolio into a new market niche. The company has produced products for the evaluation of complement activation in mouse and pig disease models. Hycult has produced four assays for each species, which will increase the availability of screening tools for complement activation in animal testing. These assays will increase our understanding of complement regulation and will thereby, indirectly, contribute to improved diagnostics in patients suffering from complement-related diseases.
11.7 Benefits for the European patients and health economy
Improved treatment modalities for ESRD patients will lead to benefits for both patients and
society. Most patients are treated with either peritoneal- or hemodialysis. Hemodialysis, in
particular, is associated with poor quality of life. A kidney transplant completely changes life for the individual ESRD patient so that he/she can return to a normal life. ESRD patients represent a major part of the societal health budget, in that they represent €40,000-80,000 of all medical spending, of which up to €15,000 is spent on hemodialysis per patient and year. An increased number of grafts available for transplantation, would dramatically improve these figures, and future use of pig kidneys or stem cell-derived kidney tissue would make it possible to cure these patients.
11.8 Training of young scientists and facilitation of exchange and mobility
PhD students and postdoctoral scientists that have been involved in the project have benefitted from project meetings, WP meetings, and other regular contacts and interactions between the partner laboratories, which has provided excellent opportunities for training them in integrated interdisciplinary techniques (e.g. materials science, immunology, innate immunity, thrombo-inflammation, transplantation, chronic kidney disease). The project has also taken advantage of the collaboration with Aegean Conferences, which is a non-profit organization with its headquarters in Philadelphia, PA, USA, but with its entire operation in Greece. The organization specializes in organizing conferences and fostering high levels of scientific dialogue. Aegean Conferences has organized two successful conferences with speakers from both academic organisations and industry.
12. Dissemination
The research results obtained by the DIREKT partners has been presented at international conferences, EU workshops and published in refereed journals. The results has also be used
in educational activities such as courses and M.Sc. Ph.D. and postdoctoral projects. The DIREKT project outcome has been disseminated to both the scientific community and the industry using the following vehicles:
12.1. Website
A specially designed website (www.KIDNEYDIREKT.org) has been an important means
of presenting the activities and the results of the consortium’s work to associated partners and other interested parties as well as to the general public. It provides information about the nature and purpose of the project, ongoing activities, a calendar of meetings and workshops, and published material such as a regular newsletter. It also provide contact details for the principal investigators in the project and for the management team.
12.2. Logotype
A project logotype has been created and used on the website and on presentations, posters, and other media in which the project is mentioned.
12.3 Publications
The results of the research activities within the project has been disseminated in the form of 126 articles in international peer-reviewed scientific journals, 130 contributions to scientific conferences, and symposia.
12.4 Review articles and Book Chapters
Seventeen review articles in high impact jounals such as Nature Reviews Nephrology, Immunology Reviews etc. concerning thromboinflammation as a pathophysiological mechanism and in the clinical setting. Eight book chapters with the same aim.
12.5 Scientific Conferences
The results of the project has been presented at scientific conferences within the fields of diabetes, immunology, transplantation, nephrology, biomaterials, tissue engineering, and cell biology. These conferences has been a great opportunity to share new knowledge with academic and industrial researchers, clinicians, and other stakeholders. More than hundred oral presentations and 5-10 posters annually have been presented.
12.6 Brochures
A consortium project brochure has been produced. It has been distributed, through the participating partners, to the scientific community and to anyone with an interest in the project.
12.7 Newsletter
An annual newsletter has been published and posted on the website. The newsletter provides updates of the project and important milestones as well as planned activities.
12.8 Meetings and Symposia
Annual meetings and open symposia have been organised in conjunction with four steering committee meetings. The consortium has also organized two open symposia (organized by Aegean Conferences) at which consortium partners and invited speakers has given presentations on ongoing research activities and preliminary findings.
12.9 Sponsored Meetings
Both meetings sponsored by Aegean Conferences were highly successful and fulfilled the specific objective of WP10 by disseminating and transferring information generated by the consortium partners. It is important to point out that in both meetings, there was attendance by conferees who are also likely to participate in other related international meetings such as Tissue Engineering and Regenerative Medicine, International Transplantation Congress, European Conference on Biomaterials, Polymeric Biomaterials, Bioengineering and Biodiagnostics. Their participation will serve to promote and disseminate the central theme of DIREKT to a much wider audience in the future. Thus, in conclusion, Aegean Conferences successfully fulfilled the Exploitation and Dissemination plan of the DIREKT consortium.
12.10 Books
A book covering multiple aspects of basic and clinical thromboinflammation was published in conjuction with the first Aegean conference meeting, authored by the speakers of the conference and edited by DIREKT (Advances in Experimental Medicine and Biology, vol. 865, 2015).
12.11 Training activities / exchange initiatives
The DIREKT project has given opportunities to have exchange of PhD students, researchers, and others to visit and/or receive training at partner universities and partner organizations. For instance the 15th European Meeting on Complement Disease was organized by the DIREKT project coordinators in Uppsala 2015 and the conference included a Teaching Day.
13. Exploitation
The overall objective of the DIREKT project has been that the results should be beneficial to society. In order to ensure that no commercial opportunities are missed, an Innovation and Exploitation Advisory Group (elected by the General Assembly) was set up at the project start. This group consisted of representatives from the participating companies, at least one academic scientist, and the Project Coordinator. The direct development of commercial products has, however, been taken care of by the appropriate company and has been performed outside DIREKT. Expected exploitation of results obtained within DIREKT but which cannot be presented due to IP issues:
a.) Already existing devices are expected to become increasingly competitive when their material surfaces are treated with the novel coatings. New devices can be produced employing materials that cannot be used today; after surface modification with the new coatings, any type of material will be suitable for use. Incorporation of the new coatings will lower costs and enable the use of materials with better-optimized properties with regard to strength, elasticity, and reproducibility.
b.) Cell surface coatings are expected to gain interest as a result of their local application in the vascular tree of the kidney graft. Both the conjugation of active biomolecules and the newly developed inhibitors have scope for exploitation and development into products. A successful local application would have a strong impact on transplantation, in that an increased number of grafts would be available, and we would most likely be able to lower the use of immunosuppression in patients receiving transplants.
c.) The regulators that have been developed may, in addition, of being part of surface coatings, also be used as systemic complement inhibiting drugs. The indications for these are broad and include autoimmune disease (SLE, RA, etc.), cardiovascular disease (cardiac infarction, stroke, etc.), transplantation and a number of yet fully identified indications such as obesity, bone fractures, sepsis etc. The total market for complement inhibitors have been estimated to be €70 billion.
14.Ethics
Ethics was taken care of by WP1 (Marianne Waern Jensen), assisted by the Management Support Team (MST). Animal ethical issues were handled by all participants, but WP1 assisted the Consortium in formulating ethical applications and verified that existing data had been approved for use in the studies. All necessary approvals for human and animal studies were collected and an independent Ethics advisor was appointed (Dr Patricia Hedenqvist, DVM, PhD) who were fully integrated within the project governance procedures. Storage of ethical approvals is the responsibility of individual participants, but copies are held by the MST and made available to the EC upon request.
In 2010, the EU adopted new regulations (Directive 2010/63/EU) based upon the “3Rs” (i.e.
Refine, Reduce, and Replace) to further improve the welfare of the animals needed for scientific purposes, and these regulations took full effect on January 1, 2013. All experiments performed on research animals have followed the EU Directive 2010/63/EU and, on a the national level, have been conducted according to ethical permissions approved in Germany by the Animal Care and Use Committee of the University Dresden, in Norway by the Norwegian Committee for Medical Research Ethics, and in Sweden by the Uppsala Ethical Committee for Animal Experimentation.
When applicable, in vitro studies were performed to replace in vivo studies. The use of newly developed whole blood models are examples of such alternative experimental strategies. Results from in vitro methods enabled reductions in the number of animals needed for experiments within the packages. These models also had the advantage that they were performed in a human environment, thereby avoiding the possible species differences that occur in large and small animals.
In WP6 the porcine experiments met all requirements for animals according to EU legislation and national SJVFS 2012:26 (L 150). The animals were provided with food and water ad libitum. Animal caretakers and veterinary nurses handled the animals during a 2-week acclimatization period, and clinical examinations was performed daily by an experienced veterinarian. The 3Rs were thoroughly considered, e.g. epidural morphine was provided to minimize pain from abdominal surgery. At the end of the experiment, after 3 months, the pigs were euthanized in their home pens with an overdose of pentobarbital sodium.
The preclinical toxicology and pharmacokinetic studies of compstatin AMY-101 (WP8) in non-human primates (NHP) were performed by contract research organizations (CRO) and was performed according to the Principles of Good Laboratory Practice (GLP). The selected CROs had permission to conduct toxicity tests in NHP under GLP conditions. The studies were performed according to the EU 2010/63 Directive and all procedures were performed taking into consideration the principles of the 3Rs. Thus, the number of animals included was as low as possible and handling procedures were strictly performed according to animal welfare legislation. A national committee for animal experiments approved the applications before onset of the studies. The result from this preclinical studies supported the assumption that a coming phase I trial would be uncomplicated and with ethical concerns.
List of Websites:
Bo Nilsson, Professor
Uppsala Universitet
Department of Immunology, Genetics and Pathology
Clinical Immunology
Email: bo.nilsson@igp.uu.se
Mobile phone: +46 70 9423977
Fax: 018-553601
Visiting address:
Dag Hammarskjölds väg 20
751 85 Uppsala
Postal address:
Rudbecklaboratoriet
751 85 UPPSALA
www.kidneydirekt.org
www.kidneydirekt.com
Chronic kidney disease is a major cause of end-stage renal disease (ESRD) worldwide: 800,000 patients in Europe and the US require long-term treatment, initially with peritoneal dialysis, and then with haemodialysis and kidney transplantation. An ESRD patient on haemodialysis has poor quality of life, with an average life expectancy of only 4 years and healthcare costs of ≈€40,000 to €80,000 per year. Kidney transplantation totally changes life for an ESRD patient, who can then return to normal life, but more widespread adoption of this treatment is still hampered by the low number of available kidney grafts. All these treatments are associated with adverse reactions that cause damaging thromboinflammation. These reactions are triggered by the intravascular innate immune system, leading to poor results and non-function.
The overall aim of this project was to clarify the mechanisms and identify the specific control points of regulation in these adverse reactions. In order to be able to significantly improve the quality of haemodialysis devices and kidney grafts, we have developed new concepts of regulation in haemodialysis and kidney transplantation. The results of this research and development have been applied in three distinctive clinical treatment modalities and in assays for the analysis of thromboinflammation in animal models:
1. We have brought a novel soluble complement inhibitor from prototype drug to a completed phase 1 clinical trial.
2. We have created nano-profiled surfaces with low activating properties that have been applied to catheters intended for haemodialysis use.
3. We have developed a PEG-lipid construct which can be used to coat biomaterial surfaces and have used it ex-vivo to coat kidney grafts intravascularly before transplantation, significantly protecting the kidney grafts against ischemia/reperfusion injury.
4. Finally, we have developed eight assays for the assessment of thromboinflammation in the mouse and pig for use in models of human disease and conditions in which thromboinflammation contributes to the pathophysiology.
All three treatment modalities are at varying stages of development into clinical treatment regimens for ESRD. When applied, we anticipate that these modalities will extend the feasible hemodialysis treatment period and, in kidney transplantation, attenuate innate immune reactions, thereby prolonging the life expectancy of the grafts and making more kidneys accessible for transplantation. The impact of these innovations is an improved quality of life and lowered cost of treatment for ESRD patients. Furthermore, all these novel techniques can be applied to other types of implantation, extracorporeal treatment, and organ transplantation and can potentially be used in the future in xenotransplantation and stem cell therapies.
Project Context and Objectives:
1. Introduction
Innate immunity is fundamental to our defense against microorganisms and foreign substances and controls the discrimination between self and non-self structures in the human body. As a consequence of its properties and actions, it is responsible for many of the incompatibility reactions that occur when foreign substances, materials, cells, and organs are introduced into the body. These reactions pose a major problem when modern biotechnological treatment modalities are used, including biomaterial devices, drug delivery systems, various bioengineered implants, cell therapies, and transplantation. Furthermore, the intravascular innate immune system can cause severe side effects in patients, as well as rejection and dysfunction of implanted devices and tissues. In this project, our focus has been on end-stage renal disease (ESRD).
Chronic kidney disease is a major cause of ESRD worldwide: 800,000 patients in Europe and in the US, respectively, require long-term treatment, initially with peritoneal dialysis, and then haemodialysis (HD) and kidney transplantation. An ESRD patient on haemodialysis has poor quality of life, with an average life expectancy of only 4 years and healthcare costs of ≈€40,000 to €80,000 per year. Kidney transplantation totally changes life for an ESRD patient, who can then return to normal life, but more widespread adoption of this treatment is still hampered by the low number of available kidney grafts.
Both HD treatment and transplantation are associated with severe adverse reactions that are caused by thromboinflammation. These reactions are triggered by the intravascular innate immune system (the complement, coagulation and contact systems), which is activated by contact between whole blood and the biosurfaces in the kidney (ischemic cells) and throughout the extracorporeal circuit (the extensive biomaterial surfaces).
HD is a life-saving treatment modality in ESRD, but it also represents a major challenge to the intravascular innate immune system, and chronic inflammation is strongly associated with cardiovascular disease (CVD) in HD. The contact between whole blood and biomaterial in the extracorporeal circuit leads to contact activation of proteins within the plasma cascade systems that results in thromboinflammation during dialysis. The contact activation initially leads to a local generation of inflammatory mediators on the material surface. This inflammation is further spread by soluble activation products and mediators (e.g the anaphylatoxins C3a and C5a, bradykinin, and various cytokines and chemokines) generated during dialysis and transported in the extracorporeal circuit back into the patient, together with activated leukocytes and platelets. The combined effect is activation of the patient’s cardiovascular endothelium, which loses its anti-thrombotic and anti-inflammatory properties leading to atherogenesis and arteriosclerosis. Because of this complex inflammatory response, it is conceivable that maximum suppression of the intravascular innate immune system is needed in order to decrease the risk for CVD in HD. This goal can be achieved either by treating the patients with systemic drugs of broader specificity than the anticoagulants used today, targeting more than one of the intravascular cascade systems, or alternatively by employing “stealth” biomaterials that cause minimal cascade system activation.
To date, kidney transplantation is the most effective treatment for patients with ESRD. Despite impressive improvements in treatment results since its introduction in the 1960s, there is still an average graft loss of 25% after 5 years and almost 50% after 10 years. The main target for improving kidney transplantation results, is a thromboinflammatory reaction known as ischemia/reperfusion injury (IRI), since ischemia followed by reperfusion evokes acute renal failure, delayed graft function, and remote organ failure. Immunosuppression does not affect the humoral innate immune system to a great extent and therefore does not attenuate the IRI. The transplantation field is also hampered by organ shortages. One option for increasing the donor pool is an increased use of marginal organs, such as those from deceased-after-cardiac-death (DCD) donors. However, these organs are also severely affected by IRI.
2. Aim
The overall aim of this project was to clarify the mechanisms of thromboinflammation and to identify its own natural specific control points of regulation in these adverse reactions in order to be able to significantly improve the quality of HD devices and kidney grafts by applying these concepts to the regulation of HD and kidney transplantation.
a.) To identify points of regulation in the cross-talk between blood (cascade systems, blood cells) and material/cell surfaces, in order to identify targets for therapeutic regulation of the intravascular innate immune system.
b.) To reduce innate immune activation by the administration of soluble molecules that inhibit the complement cascade including the major objective to to bring a prototype peptide complement factor C3 inhibitor, AMY-101, to clinical trial as a contributor to the regulation of thromboinflammation.
c.) To silence or minimize the activation of intravascular innate immune reactions by creating nano-geometrical surface profiles and regulators that can be used as coatings in devices and organs. Inhibitors that are easy to apply and applicable to a broad range of materials and cell surfaces have been constructed.
Project Results:
3. Identification of new points of regulation in thromboinflammation (WP2, WP5)
Thromboinflammation is a complex reaction that involves the intravascular innate immune system, which consists of the cascade systems of the blood (coagulation, contact, complement, and fibrinolysis systems) and cellular components such as polymorphonuclear (PMN) cells, monocytes, NK cells and platelets. The regulators of thromboinflammation that are used in the clinic today belong to the anticoagulant family, with inhibitors of thrombin and FXa as the main new members. Newly introduced inhibitors are eculizumab that blocks complement factor C5 and inhibitors of kallikrein, bradykinin receptor 2, and FXIIa. The role of the latter inhibitors has not been fully evaluated in thromboinflammation. We have focused on identifying points of thromboinflammation which involve the cross-talk between the respective cascade systems and the cellular components and investigating the possibility of using nanogeometrically modified materials in order to minimize thromboinflammatory reactions on biomaterials.
3.1a C3(H2O) as a ligand of CD11b in the formation of platelet/leukocyte complexes
The formation of complexes between PMNs and platelets is of substantial importance for the occurrence of thromboinflammation. The generation of these complexes is therefore a potential target in order to regulate thrombinflammation. In PMN-platelet complex formation, the expression of CD11b/CD18 (Mac-1) on PMNs is a prerequisite. In basic science experiments, we have identified two new ligands for CD11b which are important points of intervention in the cross-talk between PMNs and platelets. One is C3 in the form of C3(H2O), and the second is glycoprotein GPIb, both expressed on platelets. We have also further characterized developmental endothelial locus-1 (Del-1), a third ligand for CD11b with inhibitory functions, with regard to thromboinflammatory regulation.
In view of the importance of the aforementioned interactions, we employed whole-blood models and examined the effect of candidate innate immune regulators on the interactions of platelets with PMNs.
3.1a i) Formation of PMN/platelet complexes (PPC) in whole blood
As we previously reported, the formation of PPC can be triggered by thrombin receptor-activated platelets in combination with platelet-mediated complement activation. Consistent with our previous data, activation of whole blood with 33.5 µM thrombin receptor-activating peptide (TRAP)-6 led to PPC formation (CD42a-positive PMNs; Fig 1A). The conjugate formation was triggered by complement activation and was significantly inhibited by the addition of the complement (C3) inhibitor Compstatin and a C5aRA (p<0.05; n=5). Conjugate formation was also inhibited by the anti-C3a monoclonal antibody (mAb) SD17.3 (p<0.0001; n=5) as well as anti-CD11b (p<0.01; n=5) and anti-P-selectin (CD62P) (p<0.0001; n=5) mAbs. Anti-CD11b inhibited conjugate formation by 65-75%, whereas anti-C3a and anti-CD62P completely abolished PPC formation. A mAb against activated Mac-1 (CBRM1/5), which inhibited the binding by 85% (n=3), confirmed the interaction with CD11b/CD18 (Fig 1B).
3.1a ii) Complement proteins bound to platelet microparticles (PMPs)
Platelet microparticles (PMPs) were shown by flow cytometry to expose C1q, C4, and C3. The C3 results were confirmed by Western blotting using the polyclonal anti-C3c antibody. The blots showed intact alpha- and beta-chains and the C-terminal alpha-chain 45-kDa fragment, consistent with the forms of C3(H2O) previously reported on activated platelets.
3.1a iii) Formation of PMP/PMN complexes
PMPs isolated from fresh serum were added to blood-cell preparations with a reduced number of platelets and depleted of plasma proteins, and the binding of CD41a+ PMP to PMNs was analysed by flow cytometry. Upon activation with 5 nM C5a, a high percentage of the PMNs bound to CD41a+ PMPs. The PMNs bound significantly more microparticles after activation than did activated cells without added PMPs (p<0.001; n=6).
As is true for PPC formation, PMP complex formation with PMNs was also inhibited by the anti-CD11b, anti-C3a, and anti-CD62P mAbs. Anti-CD11b (p<0.01; n=9) and anti-C3a (p<0.05; n=5) mAbs significantly reduced the formation of PMP-PMN conjugates (to background levels) when compared to non-activated cells that had been incubated with PMPs. Anti-CD62P mAb lowered the PMP-PMN complex formation even further, to levels comparable to those obtained when no PMPs were added (p<0.0001; n=9). PMP-PMN complexes were unaffected by the addition of control Ig.
3.1a iv) Interaction of various forms of C3(H2O) and CD11b/CD18
The interaction between C3 and CD11b was studied with the quartz crystal microbalance with dissipation (QCM-D) technique using purified native C3 and rMac-1 (CD11b/CD18). This is a technique in which one ligand (of a putative ligand-receptor pair) is adsorbed to a sensor surface, while the other ligand is allowed to flow over the surface, enabling binding.
Native C3 was adsorbed to a hydrophobic polystyrene-coated QCM sensor surface, after which the binding of recombinant Mac-1 (rMac-1) was monitored. IgG and casein served as negative controls. The mass of bound rMac-1 to the different surfaces clearly demonstrated a binding of rMac-1 to the C3-coated surface, whereas no binding or only trace amounts of rMac-1 bound to casein and IgG, respectively. The incubation of rMac-1 with increasing concentrations of soluble iC3b prior to addition to the C3-coated surface inhibited the binding of rMac-1 at 1:1 ratio by 20%; at 5:1 by 45%, and at 10:1 by 60%. Similar experiments employing anti-CD11b mAbs showed inhibition of binding by 70-80%.
3.1b Functional testing of Del-1 in thromboinflammation
Developmental endothelial locus-1 (Del-1) was identified a few years ago as an inhibitor of leukocyte integrins, including CD11b. Given the role of CD11b in platelet-leukocyte interactions, we examined the role of the endogenous anti-inflammatory molecule Del-1 on the shaping of platelet-monocyte interactions. To this end, we assessed the effect of Del-1 on the modulation of platelet-leukocyte aggregate formation. Specifically, we employed a human whole blood model and incubated isolated islets of Langerhans. This model represents a whole-blood model of the immediate blood-mediated inflammatory reaction (IBMIR). The exposure of blood to isolated islets of Langerhans induced the activation of coagulation, as measured by the generation of thrombin-antithrombin (TAT) complexes. Moreover, the exposure of blood to islets resulted in increased formation of platelet-monocyte aggregates, a major indicator for the activation of thromboinflammation. Interestingly, the pre-treatment of blood with Del-1, prior to the addition of islets, led to decreased levels of platelet-monocyte aggregates, as well as inhibition of the coagulatory response (Fig. 2).
This heterotypic platelet-monocyte interaction was dependent on the binding of the platelet glycoprotein GPIb to the leukocyte integrin Mac-1. Moreover, we found that the N-terminal EGF-like domains of Del-1 were sufficient to block the GPIb-Mac-1 interaction.
Importantly, the presence of Del-1 was also associated with lower levels of C-peptide, thus corresponding to better islet integrity and function. Therefore, the presence of Del-1 in this whole-blood model of IBMIR substantially decreased the IBMIR-associated thromboinflammatory response. These findings clearly suggest the value of a targeted use of Del-1 for the elimination of the intravascular thrombo-inflammatory effects that are elicited upon the exposure of the host to foreign (bio)surfaces, as well as in the context of inflammatory responses to transplantation.
3.2 Activation of the lectin pathway
The lectin pathway (LP) of complement has been linked to the coagulation system in several studies, and inhibition of mannan-associated serine protease (MASP)-2 in vivo affects the coagulation system. Two proteases in the LP, MASP-1 and MASP-2, both cleave coagulation factors. MASP-1 has thrombin-like properties, while MASP-2 cleaves prothrombin to active thrombin. All these experiments have been performed in purified or semi-purified systems and no mechanistic explanation has been presented for how the LP is activated under these circumstances.
The objective of this study was to clarify the not-yet identified mechanisms involved in triggering the activation of the LP during thrombotic reactions. Novel sandwich ELISAs for the detection of complexes between MASP-1 or MASP-2 and the serine protease inhibitors (serpins) C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. Activated platelets were shown by flow cytometry to bind the recognition molecules ficolin-1, -2, and -3 (but not mannan-binding lectin [MBL]), and the binding was associated with an activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma both bind and activate MASP-1 and MASP-2. As demonstrated by ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus (SLE) patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. In conclusion, MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions both in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation. (Fig 3).
3.3 Nanostructured materials
Based on preliminary data, we hypothesized that thromboinflammation induced by biomaterials would be affected by not only the chemical nature of the material per se but also by the nanogeometry of the surface involved. Various types of nanoprofiled material surfaces (including those produced using molecular imprinting [MIP]) were produced for clarification of the fundamental conditions for complement and contact system activation on material surfaces with respect to nanogeometry as well as for potential clinical applications involving materials in contact with blood.
3.3a Production of nanostructured materials
Various nanostructured materials have been designed, fabricated, and characterized. Sparse colloidal lithography and sputter deposition were combined to produce samples on glass and silicon wafer substrates. The nanotopography was varied in terms of particle type (silica and polystyrene [PS]), particle size (34-60 nm in diameter) and inter-particle distance. Furthermore, in addition to the topographical features, all the produced materials allowed for defining various surface chemistries. The particles were analysed using nanoparticle (NP) tracking analysis prior to deposition. Optical readout for plasma protein binding was performed on glass samples using UV-visible spectrophotometry. Sets of samples were available for partners in WP2 and delivered to UU). The sets included the following sample surfaces: S1, Flat control; S2, 34 nm SiO2 NP deposited from MilliQ water; S3, 60 nm PS NP deposited from MilliQ water; S4, 60 nm PS NP deposited from 3mM NaCl, wet baked at 110°C; and S5, 60 nm PS NP deposited from NaCl, dry baked at 110°C. All sample surfaces (S1-S5) were covered with Ti and Au. The surfaces were then thiolated to obtain self-assembled monolayers (SAMs), which ensured identical surface chemistry on all sample surfaces, independent of their nanotopography. For the first set of experiments, HS-C11-OH was used. Other types of SAMs were also planned to be used but remain to be tested.
3.3b Generation of nano-geometrical profile material surfaces by applying MIP
Efforts have focused on the design, synthesis, and physical characterization of a series of polymeric nanostructures for subsequent evaluation of their impact on the complement and coagulation cascades. The primary variables being examined with respect to complement activation are morphology (with thin film-based nanowires, nano-fibers, and, as reference, various films devoid of hierarchical architectures are being studied) and the influence of inherent polymer physico-chemical properties. Our work in another project aimed at the development of protein-resistant surfaces (Norwegian Research Council, NFR) identified sulfobetaine ([2-(methacryloylamino)propyl] dimethyl(3-sulfopropyl)ammonium hydroxide) as a candidate for initial morphologic studies within DIREKT, on the basis of its “mixed-mode” characteristics (somewhat hydrophobic and zwitterionic). In parallel, work that has been accepted for publication in Biomaterials is underway to identify other polymer systems with different physico-chemical properties for the development of a series of second-generation materials. An initial technical challenge was the up-scaling of the material synthesis from QCM chip surfaces (still used here for screening for protein binding) to the glass slide format. After feedback from DIREKT partners based upon the assay results, the procedure was modified, this time including an additional reference system (prepared in a newly developed non-ionic eutectic mixture [melt]) and additional examples of each system (n=4). Also, studies based upon surfaces molecularly imprinted with heparin have been completed and published in Biomaterials Science.
3.3c Definition of the functional requirement for the recognition and activation of the complement and contact systems
A series of experiments has been done to evaluate the conditions for the recognition and activation of the complement and coagulation activation systems by the above-prepared nanostructured materials (3.3a and 3.3b) in contact with whole blood. These experiments were done by mounting the nanostructured materials onto a 2-well slide chamber model filled with fresh blood from healthy donors which was incubated with rotation for 60 min at 37°C. In one of the wells, 0.25 IU/mL heparin was added, and 0.5 IU/mL heparin was added to the other. The platelet count was measured after incubation, and no or very little loss of platelets occurred for all the materials. Plasma from all the samples was then analysed by ELISA to assess coagulation and complement activation products. All experiments were repeated at least three times.
There were clear differences in protein adsorption and blood activation between the different nanostructures generated in 3.3b (Fig 4).
For these measures, surfaces were synthesized in 3.3a with identical chemistry but different nanoprofiles. The material surfaces coated with closely packed 60-nm particles, as well as flat control surfaces, were found to be highly thrombogenic when compared to surfaces coated with sparsely packed particles of the same size or smaller. A reciprocal relationship was found regarding the initial inflammatory response; i.e. the most thrombogenic surfaces were the lowest complement activators (Fig 5).
Furthermore, huge differences in C1q binding from human EDTA-plasma were demonstrated. The surface with the smallest structure (S2, 34 nm) bound minimal amounts of C1q, i.e. like the negative control (PVC). All surfaces coated with larger NP (60 nm) bound C1q as well as did the positive control (a heparin-coated polystyrene surface), irrespective of the packing density of the NP. Coagulation Factor XII was close to or below the detection limit on all NP-bearing surfaces (S2 - S5), as well as on the negative control. The flat surface (S1) bound significant amounts of FXII, at approximately half the levels of the positive control (Fig 5).
In order to further address the question of protein adsorption on curved surfaces, we developed a novel technique for determining NP interactions with various purified proteins in solution. Particles of different sizes were first coated with a protein antigen and then with antigen-specific IgG. Finally, purified C1q was added, and the bound amount was quantified. Higher amounts of both IgG and C1q were detected on the smaller NP with a size in the same order of magnitude as the proteins, as compared to the larger particles, which have a flatter structure.
In addition, we have initiated plasma protein profiling to assess protein corona adsorbed to the various nanostructures in order to define the requirements for recognition and activation of the complement and contact systems at the protein level.
3.3d Characterization of biotin-avidin binding surfaces by applying MIP onto material surfaces
We have published a strategy for the development of biotin-avidin binding surfaces in an ‘ultra-thin film format’ (3-5 nm) in the Journal of Nanobiotechnology. The composition of the polymers was derived from earlier work using a combination of molecular dynamics-based studies of potential pre-polymerization mixtures and radioligand studies of the corresponding bulk polymers. Evaluation of ligand-biotin selective polymer film recognition properties was undertaken using QCM, and appropriate control systems were employed.
The biotinyl moiety-selective molecularly imprinted polymer-grafted SAM-Au/quartz nano-films showed significant selectivity for biotinyl-bearing carbohydrate and peptidic structures and could efficiently distinguish the biotinylated structures from the native counterparts (Fig 6, upper panel). The interaction of the analyte with imprinted sites through the formation of affinity complexes was determined by calculating the apparent stability constant (Ks=ka/kd) and showed a two-fold higher stability for binding to the MIP than to the reference (Fig 6, lower panel).
Because of the frequent problems we encountered with the mechanical stability of the polymer films, we devoted our efforts to developing the use of silanized surfaces and organic modifiers (low concentrations of organic solvents) to enhance the yield from the fabrication process. A series of second generation of biotinyl moiety imprinted materials have been synthesized in two formats, ultra-thin film and with hierarchical architectures derived from sacrificial meso-structure (mono-disperse polystyrene). The modified surfaces have been characterized by reflection absorption infrared spectroscopy (RAIRS), scanning electron microscopy (SEM), atomic force microscopy (AFM), ellipsometry, and X-ray photoelectron spectroscopy (XPS).
4. Development of regulators of thromboinflammation to be used in hemodialysis and kidney transplantation (WP3, 4, 5)
One approach to regulating thromboinflammatory reactions on biosurfaces in contact with whole blood (such as occur during haemodialysis on the biomaterial surfaces and in transplantation on cell surfaces during IRI) is to precoat the surface with a non-reactive shield or a regulator of the individual cascade systems or cells. We have investigated a number of alternative coatings that use different mechanisms of regulation and have identified some of them as candidates for further development into clinically applicable treatment modalities. For instance, we have developed various types of PEG and heparin derivatives for both direct protection of biosurfaces and also to be used as linkers of protective peptides and macromolecules to biosurfaces.
4.1. PEG coatings
4.1a. Coating of cell surfaces with a PEG-lipid construct
Evaluation of different PEG constructs on various cell surfaces was performed. Human mesenchymal stromal cells (MSCs) were coated with a PEG-phospholipid construct and showed less platelet consumption and a decrease in activation of the coagulation and complement systems when tested in an in vitro whole blood model. These findings were confirmed after coating of hepatocytes with PEG (Fig 7). This promising inhibitor of thromboinflammation was further tested in small and large animal models and the intension is to bring this technique to the clinical transplantation. See also 8.2.
4.1b. Development of material-coating techniques with PEG and heparin
In addition, we developed material-coating techniques with PEG. For instance, Pluronic F108 was adsorbed to hydrophobic surfaces (such as PS and PVC), resulting in diminished activation of the coagulation and complement systems. Efforts were also focused on the development of a thicker coating that could better shield the surface than did Pluronic F108, thereby more thoroughly decreasing the biomaterial-induced adverse effects. To this end, Pluronic molecules were polymerized using multi-armed PEG molecules covalently attached to the Pluronic. This conjugation approach resulted in further decreased activation of both the coagulation and complement pathways.
Also, a heparin-conjugate and unfractionated heparin were attached through heparin-binding peptides (HBPs) conjugated to Pluronic F108 that was adsorbed to a material surface. This approach resulted in diminished activation of the coagulation and complement systems. Alternatively, we used four-arm PEG-maleimide, to which HBPs was attached (Fig 7). Heparin-conjugate or unfractionated heparin (UFH) was then captured on the surface by peptides. Also, a heparin conjugate was designed by covalently attaching unfractionated heparin to multi-arm PEG, which was immobilized on the surface that was pre-coated with Pluronic-HBP. This composite was achieved either by first attaching the eight-arm PEG to the Pluronic and then adding UFH or by first making the eight-arm PEG+UFH conjugate and attaching it to the surface with Pluronic-HBP. Both approaches were efficient, as shown by heparin quantification.
4.2. Factor H-like inhibition
Factor H (FH) within the alternative pathway (AP), is one of the most powerful natural inhibitors of complement activation. Within DIREKT, two strategies have been developed to engage this inhibitor. The first principle was to use a peptide with affinity for FH to pull down the inhibitor to the biosurface. The second was to truncate the FH gene so that only the active sites in the N- and C-terminal parts of FH were expressed, with the resulting molecule being designated “mini-FH”. We have achieved significant progress in the development of the peptide 5C6 and the complement inhibitor mini-FH, which are among our main deliverables.
4.2a. Evaluation of the 5C6 molecule
The 5C6 molecule, a peptide with the ability to bind and recruit FH to sites of complement activation, was developed and tested in a mouse model of arthritis.
The peptide 5C6 showed high selectivity for FH and binds FH from different species, i.e. human, monkey, pig, rat, and mouse. This finding was particularly important in the context of the DIREKT project, given the inclusion of mouse and pig models in various projects. Further, we have established a surface plasmon resonance (SPR)-based assay and quantified the monovalent binding affinity of 5C6 to FH with a KD of ~150 nM. This assay was also used for the screening of an alanine scanning library of 5C6, revealing that several amino acids in the cyclic part and at the C-terminus, but not in the N-terminus of the peptide, drive the interaction with FH. Using site-specific truncation and de-novo synthesis, we showed that truncated analogs lacking the N-terminus have comparable activities as the full-length peptide. Because of its shorter size and more effective synthesis, we therefore selected truncated 5C6 as our new lead compound. Furthermore, using the SPR assay, we narrowed down the binding region of 5C6 to FH8-15. Apart from the molecular and in vitro studies, we conjugated 5C6 with a peptide able to bind the synovial membrane and used this construct in a mouse model of antigen-induced arthritis. Our data showed that the dual peptide was able to bind to inflamed synovium in vivo and recruit FH to the inflammation sites, inhibiting swelling and migration of inflammatory cells in the arthritic mice.
4.2b. Mini-FH inhibitor
Regarding the mini-FH inhibitor, we showed that it maintains or exceeds the host-cell protective activity of FH despite its 70% reduction in size. Human mini-FH showed inhibitory activity in pig plasma in collaboration with WP8, supporting the suitability of mini-FH for in vivo studies. Furthermore, our pharmacokinetic studies performed both in mice and non-human primates revealed that although a rapid initial clearance of the inhibitor after intravenous injection, residual protein could be detected much later, indicating longer cell/tissue residence when compared to the circulatory half-life. Several collaborative animal experiments have been initiated for human mini-FH, e.g. with WP5. A structural comparison of several regulator homologs bound to C3b has been published, revealing several determinants that define complement-inhibitory activity and adding useful insights for the design of next-generation inhibitors. In addition, other FH variants, i.e.; FHL-1, FHΔ10-15, and midi-FH, were engineered, expressed, and tested to evaluate their ability to inhibit the deposition of C3b triggered by the lipopolysaccharide (LPS)- or paroxysmal nocturnal hemoglobinuria (PNH) cell-induced AP. This comparison indicated that FHL-1 is responsible for about one-third of the systemic complement regulation of the AP, a finding that has implications for the pathophysiology of diseases associated with genetic variations within the FH protein family. It also showed that the binding affinity for C3b is correlated with the number of potential binding sites, and that FHΔ10–15 and midi-FH are substantially more active AP regulators than is mini-FH, in assays employing LPS but not PNH cells, suggesting that the mechanism of action of these proteins is subject to different limitations when they are acting on host/self versus foreign (or artificial) surfaces (Fig 8).
4.3. C1s inhibitor
Apart from AP inhibitors, we also recombinantly expressed the classical pathway (CP) inhibitor BD001 in yeast (P. pastoris) and revealed strong binding affinity for C1s and CP-inhibitory activity in human plasma. Unfortunately, BD001 development was hampered by the heterogeneity of the yeast-derived product. We then attempted to express the protein in expression systems that facilitate folding (BD001 has 20 cysteines) and allow for proper post-translational modification (sulfation contributes to its overall activity). The use of mammalian cells (HEK293T) resulted in expression but no secretion of the protein. A baculovirus system was also evaluated with two different cells (SF9, Hi5), but the desired protein was not expressed. In the meantime, a group from Australia was able to express BD001 (Gigastasin) in High Five insect cells in large enough quantities to resolve the crystal structure of the inhibitor in complex with C1s. Gigastasin appears to block the active sites of C1s and MASP-2 and was able to inhibit the activation of the CP and the LP pathways, supporting the suitability of BD001 for use as a complement inhibitor.
4.4. C4BP-peptides
We also characterized several peptides, i.e. P1, CKENQGKLEKLELDYLKKL; P2, CDKAMKAKVATYLGGLPDVPFSAT; P3, CSGIAQFHIDYNNVSSAEGWHVN; and P4, CGVQLDLDEAI in terms of their ability to recruit C4BP and inhibit CP activation. These peptides were all conjugated to maleimide-PEG-lipid and used to coat cell surfaces. P1, P2, and P3 were considered potential therapeutic candidates, since they showed binding to C4BP by QCM-D; in contrast, only minor C4BP recruitment was achieved using P4.
5. Development of assays for assessment of mouse and porcine complement (WP5 and 6)
The cascade systems of the blood have traditionally been investigated in humans and in human blood. Assays to assess the activation of the systems in other species have been difficult to obtain. DIREKT partner Hycult Biotech has developed assays for complement analyses in mouse and pig, the most common mammals for in vivo research.
Development of immunoassays to detect activation of the mouse complement system was a main objective of WP5. Partner Hycult Biotech developed four different assays for the assessment of specific activation products and pathways of complement system, including detection of activation of the CP, AP, and LP. All assays successively passed quality control and testing within the consortium.
The deliverable was to establish three functional complement pathway assays (for the CP, AP and LP) and an assay for the porcine activation fragment C5a. All four assays were successfully established and met all the requirements for assay performance and quality control, as determined according to internal standards. Results from independent validation experiments showed that assay performance is good and that the achieved data are accurate and reliable. The CP and LP assays are now commercially available; the AP and the C5a assays are expected to become available early in 2018 (Fig 9).
6. Testing of inhibitors of thromboinflammation in vitro and in small animals (WP5)
In order to further evaluate promising regulators of thromboinflammation that are candidates for further development and in vivo large animal testing, the treatment modalities were tested in vitro and in vivo in small animal models.
6.1. Inhibition of thromboinflammation in IBMIR
We tested the developed inhibitors of intravascular innate immunity using in vitro and small animal models. The effect of innate immune regulators on the modulation of thrombo-inflammatory reactions was studied by establishing whole-blood models. We focused on the instant blood-mediated inflammatory reaction (IBMIR), which is elicited upon islet transplantation (Tx) and causes islet dysfunction. We used a model in which human whole blood is incubated with isolated porcine islets of Langerhans and a model in which whole blood is in contact with porcine endothelium; both models elicit an IBMIR-like inflammatory reaction, including a strong thrombo-inflammatory response. In this context, we studied the role of endogenous anti-inflammatory agents or highly specific complement inhibitors as possible therapeutic interventions for thrombo-inflammatory reactions (including leukocyte adhesion, leukocyte-platelet interactions, and activation of coagulation). For instance, Del-1 was tested in these models and was found to be a promising inhibitor of thromboinflammation. Using these models, we focused our activities on the immobilization of regulators on cellular surfaces and (bio)surfaces, including regulators of the complement system such as apyrase, a regulator of platelet and coagulation activation, and heparin-binding peptides. Importantly, co-immobilization of regulators on the surface of different target cells led to attenuated thrombo-inflammatory responses upon exposure of these cells to human blood.
6.2. Coating endothelial cells with heparin
A conjugate of multiple unfractionated heparin chains coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC (a conjugate of 70 heparin molecules linked to a polymer chain) to protect wild-type (WT) and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNF-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation, and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNF-treated PAEC with CHC (100µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged the clotting time of human blood (115.0±16.1min p<0.001) when compared to WT PAEC (34.0±8.2min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3±11.3min p<0.001; GTKO.hCD46.hTBM: 146.2±20.4min p<0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.
To further study the effect of the heparin coating on the regulation of the adverse effects caused by exposure of the host to (bio)surfaces, we established a mouse Tx model of ischemia-reperfusion injury and a mouse model of islet Tx. We studied the function of heparin coating and of complement regulators. Of note, CHC improved the function of the graft in mice that received a kidney transplant after cold storage; this effect was accompanied by inhibition of inflammation. Together, these data suggest that single or combined use of innate immune regulators coated on biosurfaces may be advantageous for the elimination of the thrombo-inflammatory effects elicited upon exposure of the host to foreign (bio)surfaces.
7. Testing of inhibitors of thromboinflammation in large animal models (WP6)
Pig models of kidney transplantation were used to evaluate selected promising inhibitors of thromboinflammation, with the aim of abrogating the host response to the transplanted organ with regard to intravascular innate immunity and IRI.
7.1 In vitro and ex vivo models of kidney IRI and antibody-mediated rejection
Our specific objective here was to establish ex vivo models to study pathophysiology related to kidney transplantation and to apply regulators to dampen the host response. Two complementary models were established, one based on cultured endothelial cells (EC) in vitro, and another on isolated ex vivo porcine kidneys. The aim was to characterize and apply regulators to these model systems under conditions of IR and antibody-mediated rejection (AMR). In the cell-based assay, hypoxia and subsequent reperfusion with fresh human whole blood mimicked IRI; inclusion of antibodies against EC simulated AMR. IR in isolated porcine kidneys was accomplished by overnight cold storage of the explanted organ and subsequent reperfusion with autologous blood. In separate experiments, fresh human whole blood was employed to trigger a xenoreaction simulating AMR. Complement activation was a major readout in the EC model; a broad panel of thromboinflammatory read-outs and markers of kidney injury and function were characterized in the isolated organ model. Regulators, primarily PEG-lipid and mini-FH, were administered in the models. The main findings were that PEG-lipid lowered platelet consumption in AMR of isolated kidneys. The sampled biological material from the mini-FH study are currently under analysis.
8. Three main potential clinical treatment modalities resulting from DIREKT project activities
8.1 Development of the C3 inhibitor AMY-101 from prototype to in-human drug
The initial focus of Amyndas in the DIREKT was to bring AMY-101 (of the Compstatin family) from a preclinical drug candidate to the stage where it can be tested in a clinical trial with the indication of ABO-incompatible kidney transplantation.
Our initial focus in the DIREKT project was on the design and conduct of the appropriate pre-clinical studies to evaluate the safety of the C3 complement inhibitor AMY-101 and its pharmacokinetic (PK) profile before its application in human clinical trials. Therefore, we designed our preclinical strategy and proceeded to the manufacturing of preclinical grade AMY-101 and the conduct of GLP toxicology studies. The preclinical toxicology studies in non-human primates showed that AMY-101 did not show any systemic toxicity after repeated administration, and the GLP PK study showed a good PK profile, which can be used to guide the human clinical studies. In the meantime, GMP grade (clinical grade) AMY-101 was produced, to be used in the human clinical studies and long-term stability studies were initiated to evaluate the stability over time.
In Q1 2017 Amyndas submitted a clinical trial application for the first in human (phase I) clinical study to the US FDA. The FDA approved the application while in the meantime, the stability studies confirmed long-term stability of AMY-101. Following FDA approval, Amyndas started the phase I clinical trial in human volunteers and successfully completed the study in November 2017.
This study involved testing AMY-101 in human volunteers, to establish the safety and PK/PD profile of the drug. The study comprised of a single ascending dose (SAD) part and a multiple dose (MD) part. In the SAD part of the study single doses of AMY-101 were administered systemically through the subcutaneous and intravenous routes, and followed for 21 days of monitoring. In the MD part of the study multiple doses of AMY-101 were administered by subcutaneous administration, and the subjects were followed for 14 days after last dosing. AMY-101 was well tolerated in both the SAD and MD part of the study. No serious adverse events of AMY-101 have been observed in any of the subjects dosed. Analysis of the PK/PD profile showed that saturation of target C3 is reached after one administration at the pharmacologically active dose, and this saturation can be maintained with daily dosing at this level.
At the same time Amyndas prepared the phase 2a clinical study protocol,l for the application of AMY-101 in ABO incompatible kidney transplanted patients.
In summary, during the duration of the DIREKT project, all the objectives and milestones of WP8 have been successfully completed.
8.2 Demonstration of the potential clinical benefit from PEG-lipid coating with regard to thromboinflammation (IRI) in a porcine in vivo allogenic transplantation model
The objective was to establish a porcine in vivo survival model of kidney transplantation, to characterize the model, and to apply a regulator to evaluate its effect on kidney function and survival (task and deliverable). The model, which was established at month 12 (a milestone), is based on explantation of kidneys from a donor pig, cold preservation of the explanted kidneys, and subsequent transplantation into a swine leucocyte antigen (SLA )-mismatched recipient, both of whose native kidneys had been removed. The recipients were under continuous surveillance, were clinically examined regularly, and blood, urine, and tissue samples were regularly obtained. Plasma, urine, and tissue were examined for a broad panel of thromboinflammatory read-outs, and markers of kidney injury and function were measured. The PEG-lipid was extensively evaluated in this model, and the regulator was then administered to the explanted kidneys closely immediately before transplantation. Donors who received PEG-lipid-treated kidneys had lower levels of complement and coagulation activation, as compared to vehicle-treated controls. Pro-inflammatory cytokines (IL-1beta, IL-6) were also lower, and kidney function was improved. The intension is now to bring this technique to the clinic.
8.3 Development of a haemodialysis catheter, applying novel knowledge concerning the effect of nanogeometry on thrombinflammation
As a clinical application of the studies of the effect of the nanogeometry on thromboinflammation, we successfully developed fabrication approaches to coat multiple catheter materials with nanostructures and tested the functionality of the coatings to reduce blood activation and bacterial adhesion. Nanostructuring approaches were developed to prepare pure nanotopographic profiles (30 to 140-nm profiles were prepared) or multifunctional coatings containing nanotopographic profiles, noble metal chemistries, and anti-coagulants. These coatings were assessed on catheter materials and on commercial catheters. These prototype coatings on commercial catheter materials were tested for blood compatibility (using the Chandler loop test) and bacterial adhesion (using the Ahearn test). Several multifunctional coatings showed significant reductions in blood activation (TAT level, blood platelet count, C3a level, and sC5b-9 level) and bacterial adhesion when compared to the same commercial catheters in uncoated form.
A coating was developed which significantly reduced both blood activation (by >95%) and bacterial adhesion (by >90%) when compared to the same commercial catheter, uncoated. This coating is now intended to be CE-marked and commercialised for haemodialysis catheters.
9. Exploitation and dissemination (WP10 and WP11)
Other objectives of DIREKT were the transferral of information and the identification of a series of questions related to ESRD with regard to academic and industrial issues. A number of activities were carried out to achieve these goals.
9.1. Aegean Conferences on Biosurfaces (WP10)
The partners of the Aegean Conferences have developed expertise during the past 18 years in the organization of scientific conferences. Over the years, these conferences have fostered dissemination, training, and scientific dialogue in disciplines related to the central theme of the DIREKT consortium.
In accordance with the Exploitation and Dissemination plan of the DIREKT consortium, Aegean Conferences sponsored two conferences under the titles ‘1st and 2nd International Conferences on Immune Responses to Biosurfaces: Mechanisms and Therapeutic Interventions’. The first conference took place from September 27 to October 2, 2014 in Chania, Greece, and the second from September 26 to October 1, 2016 in Rhodes, Greece (Fig 10). In both of these face-to-face meetings, explorations of innovations and inventions were exchanged among all of the WP leaders (and other representatives of the consortium) and leaders of industries dealing with ESRD-related treatment modalities. The conference programs can be found on first conference and second conference. Collectively, these meetings were attended by over 100 participants, including the DIREKT partners from academia and industry as well as the members of the Advisory Board of DIREKT, who presented their progress. In addition, WP-focused discussions took place during and after the meetings. As is typical of the style of the Aegean Conferences, there were several awards conferred on young trainees who presented their work during these conferences.
9.2 Publication of conference proceedings
Directly relevant to the objectives of WP10 is the publication of the proceedings of the first conference that appeared in Advances in Experimental Medicine and Biology (vol. 865, 2015).
9.3 Dissemination and exploitation (WP11)
The DIREKT project has been very productive in terms of dissemination and exploitation activities, generating 126 per-reviewed publications, one academic PhD thesis, 130 dissemination activities (such as oral presentations and posters at international conferences and scientific meetings), and 8 exploitable foregrounds during the 4 years of the consortium. It has also established a large number of fruitful collaborations among the partners involved, in both academia and industry, that will be continued after the project period ends.
10. Conclusion
We have clarified several mechanisms and identified specific control points of regulation in adverse reactions caused by thromboinflammation and in order to be able to significantly improve the quality of haemodialysis devices and kidney grafts we have developed new concepts of regulation in haemodialysis and kidney transplantation. The results of this research and development have been applied in three distinctive clinical treatment modalities and in assays for analyses of thromboinflammation in animal models:
1. We have brought a novel soluble complement inhibitor from prototype drug to a completed phase 1 clinical trial.
2. We have created nano-profiled surfaces with low activating properties that have been applied to catheters intended for haemodialysis use.
3. We have developed a PEG-lipid construct which can be used to coat biomaterial surfaces and have used it ex-vivo to coat kidney grafts intravascularly before transplantation, significantly protecting the kidney grafts against ischemia/reperfusion injury.
4. Finally, we have developed eight assays for the assessment of thromboinflammation in the mouse and pig for use in models of human disease and conditions in which thromboinflammation contributes to the pathophysiology.
All three treatment modalities are at varying stages of development into clinical treatment regimens of ESRD. When applied we anticipate that these modalities will extend the feasible hemodialysis treatment periods and in kidney transplantation attenuate innate immune reactions thereby prolonging the life expectancy of the graft and make more kidneys accessible for transplantation. The impact of this is improved quality of life and lowered costs for treatment of ESRD patients. All the novel techniques can be applied to other types of implantations, extracorporeal treatments and organ transplantations and in the future potentially be used in xenotransplantation and stem cell therapies.
Potential Impact:
11. Impact
11.1 New treatment strategies and improved health
The innate immune system of the blood and other tissue fluids represents a serious obstacle to the development of treatment modalities using tissue transplants and device implants, since it provoke thromboinflammatory reactions. The focus of this project has been the chronic kidney disease known as end-stage renal disease (ESRD).
a.) In hemodialysis patients, the frequent treatments trigger side effects of whole-body thromboinflammation, leading to a dramatically increased risk for cardiovascular disease and for infections. Regulation of the complement, contact and coagulation systems activation, and other aspects of innate immunity will reduce adverse effects in these patients and lower the risk of arteriosclerosis, thrombosis and in severe infections. Today, in their attempts to control the intravascular innate immune system, manufacturers of biomedical device implants are almost entirely restricted to an empirical selection of compounds for biomedical device design.
Development in DIREKT of active easy-to-apply coatings and optimal nanostructured geometries to control immune activation will therefore be of great significance and make it possible to create biomedical devices built of materials that meet the necessary requirements for stability, abrasion, and strength, independent of their inherited properties to trigger thromboinflammation.
b.) Successful transplantation has an immense impact on the quality of life and normalizes the life and health of the patients. The intravascular innate immune system causing thromboinflammation, is involved in various types of rejection during, immediately after, and in the long term after kidney transplantation. This rejection has severe consequences, such as limiting the selection of grafts for specific recipients and reducing the survival rate of the transplanted grafts. The results of DIREKT using soluble complement inhibitors and cell coatings will improve the quality of life for patients with ESRD by optimizing the conditions for kidney transplantation, with the aim of increasing the number of patients that can regain a normal life. In the future, techniques developed within this program may also prove applicable to kidney xenotransplantation (and stem cell therapies), representing a large step forward toward a greater availability of kidney grafts and transplant-related restoration of normal life for ESRD patients.
c.) Also, patients with other diseases such as cardiovascular disease (those receiving stents, cardiopulmonary bypass, heart transplantation) and liver disease (liver dialysis and transplantation), in which transplantation and extracorporeal treatment are important treatment modalities, will benefit greatly from the project.
11.2 A new soluble complement inhibitor (AMY-101)
The phase 1 clinical trial of AMY-101 performed by partner Amyndas is a great achievement and step forward which will greatly promote the development of this compound as an immunoregulatory drug for use in kidney transplantation. AMY-101 has high potential in that it would be a novel inhibitor to target C3 in the complement cascade. Eculizimab, the only clinically available complement inhibitor which targets C5, acts further downstream than AMY-101. Therefore, AMY-101 has a broader effect, inhibiting not only C5a and the terminal complex C5b-9 but also effects mediated by C3 fragments.
The ABO incompatibility barrier is a severe obstacle to transplantation, particularly for living donor transplantations. Recent studies reveal that complement is instrumental in preventing ABO incompatible organ rejection during transplantation. ABO-incompatible kidney transplantation is a possible indication for AMY-101, since it is expected that AMY-101 can make non-matched donor transplantations safer and can lead to accommodation of the graft. This would be of great benefit for patients with ESRD as it could lead to an increased accessibility of donor organs and it would facilitate an increase of the number of transplantations for patients in need.
11.3 Coatings for protection of cells against thromboinflammation
The newly developed coatings of cell surfaces have been shown to significantly protect cells against attack from the innate immune system. In transplantation, the condition primary-non-function is associated with poor long-term survival of the kidney graft and is triggered by the thromboinflammatory reaction called ischemia/reperfusion injury (IRI). The developed coatings lower IRI and are therefore expected to improve the life span of the graft thereby improving quality of life for the patient, increase the number of available graft and lower the cost of the ESRD treatment.
11.4 New bioactive nano- and microstructured materials
The area of nano- and microstructured materials has become a prioritized research direction in Europe, through integrated efforts in FP5, 6 and 7 and in the Nanomedicine European Technology Platform. These efforts have developed knowledge, tools, and frameworks for understanding and design of nanprofiled structures. In DIREKT, our close partnership and integrated approach provides a strong basis for innovation. We have developed robust fabrication routes that can be scaled up to industrial-level processes, enabling the testing of nanostructured materials in medical devices.
a.) One example of this is the new coating developed by the DIREKT partner Bactiguard in collaboration with Århus and Uppsala Universities. This coating combines Bactiguards previous coatings with low thrombinflammatory properties. This will lower the thromboinflammatory reactions in vivo. The surface treatment can potentially also be used in other extracorporeal applications when whole blood comes in contact with biomaterials e.g. heart pumps, cardiovascular bypass etc. The clinical application of this coating is in the near future considering that CE marking is already obtained.
b.) The newly developed protocol based on the findings in DIREKT that will apply nanostructured surfaces in the functionalization of polymer surfaces with artificial avidin, could constitute a potential breakthrough in the field of biomaterial compatibility and would offer a very versatile procedure for adapting surfaces for specific biological challenges.
11.5 New assays for assessment of thrombinflammatory parameters in animal models
Mouse and pig are among the most common and versatile animals for studies of human disease. Partner Hycult has developed four assays for each species. These new assays will replace the previous time consuming and laborious tests, thereby making studies focusing on thromboinflammatory reactions in vivo, more accessible.
11.6 Reinforcement of European SMEs
The four companies that have been involved in DIREKT, has worked closely together with the academic scientists on this project in order to help patients with ESRD to achieve an improved life quality through improved hemodialysis and kidney transplantation. An EU project like DIREKT has also enable the development of personal relations with other participating companies and universities, an invaluable advantage for start-up companies seeking to secure future business relations.
a.) To that end, Amyndas Pharmaceutical, a Greek biotechnology company spun out from the University of Pennsylvania was founded in order to transfer technology from the US to Europe, specifically to Greece. The company develops therapeutics for inflammatory diseases and disorders based on patented technology for modulating the complement system. This company has licensed technology from the University of Pennsylvania and translated it into the clinically applicable AMY-101 for threatment of among other indications, patients with ESRD. This project has been a crucial step for Amyndas in developing a commercial platform for drugs against complement-mediated diseases.
b.) Hycult Biotech is a Dutch SME specialized in the development, manufacturing and commercialization of research reagents (antibodies, immunoassays) in the field of innate immunity. They develop products related to complement, antimicrobial peptides, acute phase proteins, TLR, LPS and microbial toxins. Hycult Biotech further has a specific interest in inflammation and cell - and tissue damage. The DIREKT project has provided a unique opportunity to extend the company’s product portfolio into a new market niche. The company has produced products for the evaluation of complement activation in mouse and pig disease models. Hycult has produced four assays for each species, which will increase the availability of screening tools for complement activation in animal testing. These assays will increase our understanding of complement regulation and will thereby, indirectly, contribute to improved diagnostics in patients suffering from complement-related diseases.
11.7 Benefits for the European patients and health economy
Improved treatment modalities for ESRD patients will lead to benefits for both patients and
society. Most patients are treated with either peritoneal- or hemodialysis. Hemodialysis, in
particular, is associated with poor quality of life. A kidney transplant completely changes life for the individual ESRD patient so that he/she can return to a normal life. ESRD patients represent a major part of the societal health budget, in that they represent €40,000-80,000 of all medical spending, of which up to €15,000 is spent on hemodialysis per patient and year. An increased number of grafts available for transplantation, would dramatically improve these figures, and future use of pig kidneys or stem cell-derived kidney tissue would make it possible to cure these patients.
11.8 Training of young scientists and facilitation of exchange and mobility
PhD students and postdoctoral scientists that have been involved in the project have benefitted from project meetings, WP meetings, and other regular contacts and interactions between the partner laboratories, which has provided excellent opportunities for training them in integrated interdisciplinary techniques (e.g. materials science, immunology, innate immunity, thrombo-inflammation, transplantation, chronic kidney disease). The project has also taken advantage of the collaboration with Aegean Conferences, which is a non-profit organization with its headquarters in Philadelphia, PA, USA, but with its entire operation in Greece. The organization specializes in organizing conferences and fostering high levels of scientific dialogue. Aegean Conferences has organized two successful conferences with speakers from both academic organisations and industry.
12. Dissemination
The research results obtained by the DIREKT partners has been presented at international conferences, EU workshops and published in refereed journals. The results has also be used
in educational activities such as courses and M.Sc. Ph.D. and postdoctoral projects. The DIREKT project outcome has been disseminated to both the scientific community and the industry using the following vehicles:
12.1. Website
A specially designed website (www.KIDNEYDIREKT.org) has been an important means
of presenting the activities and the results of the consortium’s work to associated partners and other interested parties as well as to the general public. It provides information about the nature and purpose of the project, ongoing activities, a calendar of meetings and workshops, and published material such as a regular newsletter. It also provide contact details for the principal investigators in the project and for the management team.
12.2. Logotype
A project logotype has been created and used on the website and on presentations, posters, and other media in which the project is mentioned.
12.3 Publications
The results of the research activities within the project has been disseminated in the form of 126 articles in international peer-reviewed scientific journals, 130 contributions to scientific conferences, and symposia.
12.4 Review articles and Book Chapters
Seventeen review articles in high impact jounals such as Nature Reviews Nephrology, Immunology Reviews etc. concerning thromboinflammation as a pathophysiological mechanism and in the clinical setting. Eight book chapters with the same aim.
12.5 Scientific Conferences
The results of the project has been presented at scientific conferences within the fields of diabetes, immunology, transplantation, nephrology, biomaterials, tissue engineering, and cell biology. These conferences has been a great opportunity to share new knowledge with academic and industrial researchers, clinicians, and other stakeholders. More than hundred oral presentations and 5-10 posters annually have been presented.
12.6 Brochures
A consortium project brochure has been produced. It has been distributed, through the participating partners, to the scientific community and to anyone with an interest in the project.
12.7 Newsletter
An annual newsletter has been published and posted on the website. The newsletter provides updates of the project and important milestones as well as planned activities.
12.8 Meetings and Symposia
Annual meetings and open symposia have been organised in conjunction with four steering committee meetings. The consortium has also organized two open symposia (organized by Aegean Conferences) at which consortium partners and invited speakers has given presentations on ongoing research activities and preliminary findings.
12.9 Sponsored Meetings
Both meetings sponsored by Aegean Conferences were highly successful and fulfilled the specific objective of WP10 by disseminating and transferring information generated by the consortium partners. It is important to point out that in both meetings, there was attendance by conferees who are also likely to participate in other related international meetings such as Tissue Engineering and Regenerative Medicine, International Transplantation Congress, European Conference on Biomaterials, Polymeric Biomaterials, Bioengineering and Biodiagnostics. Their participation will serve to promote and disseminate the central theme of DIREKT to a much wider audience in the future. Thus, in conclusion, Aegean Conferences successfully fulfilled the Exploitation and Dissemination plan of the DIREKT consortium.
12.10 Books
A book covering multiple aspects of basic and clinical thromboinflammation was published in conjuction with the first Aegean conference meeting, authored by the speakers of the conference and edited by DIREKT (Advances in Experimental Medicine and Biology, vol. 865, 2015).
12.11 Training activities / exchange initiatives
The DIREKT project has given opportunities to have exchange of PhD students, researchers, and others to visit and/or receive training at partner universities and partner organizations. For instance the 15th European Meeting on Complement Disease was organized by the DIREKT project coordinators in Uppsala 2015 and the conference included a Teaching Day.
13. Exploitation
The overall objective of the DIREKT project has been that the results should be beneficial to society. In order to ensure that no commercial opportunities are missed, an Innovation and Exploitation Advisory Group (elected by the General Assembly) was set up at the project start. This group consisted of representatives from the participating companies, at least one academic scientist, and the Project Coordinator. The direct development of commercial products has, however, been taken care of by the appropriate company and has been performed outside DIREKT. Expected exploitation of results obtained within DIREKT but which cannot be presented due to IP issues:
a.) Already existing devices are expected to become increasingly competitive when their material surfaces are treated with the novel coatings. New devices can be produced employing materials that cannot be used today; after surface modification with the new coatings, any type of material will be suitable for use. Incorporation of the new coatings will lower costs and enable the use of materials with better-optimized properties with regard to strength, elasticity, and reproducibility.
b.) Cell surface coatings are expected to gain interest as a result of their local application in the vascular tree of the kidney graft. Both the conjugation of active biomolecules and the newly developed inhibitors have scope for exploitation and development into products. A successful local application would have a strong impact on transplantation, in that an increased number of grafts would be available, and we would most likely be able to lower the use of immunosuppression in patients receiving transplants.
c.) The regulators that have been developed may, in addition, of being part of surface coatings, also be used as systemic complement inhibiting drugs. The indications for these are broad and include autoimmune disease (SLE, RA, etc.), cardiovascular disease (cardiac infarction, stroke, etc.), transplantation and a number of yet fully identified indications such as obesity, bone fractures, sepsis etc. The total market for complement inhibitors have been estimated to be €70 billion.
14.Ethics
Ethics was taken care of by WP1 (Marianne Waern Jensen), assisted by the Management Support Team (MST). Animal ethical issues were handled by all participants, but WP1 assisted the Consortium in formulating ethical applications and verified that existing data had been approved for use in the studies. All necessary approvals for human and animal studies were collected and an independent Ethics advisor was appointed (Dr Patricia Hedenqvist, DVM, PhD) who were fully integrated within the project governance procedures. Storage of ethical approvals is the responsibility of individual participants, but copies are held by the MST and made available to the EC upon request.
In 2010, the EU adopted new regulations (Directive 2010/63/EU) based upon the “3Rs” (i.e.
Refine, Reduce, and Replace) to further improve the welfare of the animals needed for scientific purposes, and these regulations took full effect on January 1, 2013. All experiments performed on research animals have followed the EU Directive 2010/63/EU and, on a the national level, have been conducted according to ethical permissions approved in Germany by the Animal Care and Use Committee of the University Dresden, in Norway by the Norwegian Committee for Medical Research Ethics, and in Sweden by the Uppsala Ethical Committee for Animal Experimentation.
When applicable, in vitro studies were performed to replace in vivo studies. The use of newly developed whole blood models are examples of such alternative experimental strategies. Results from in vitro methods enabled reductions in the number of animals needed for experiments within the packages. These models also had the advantage that they were performed in a human environment, thereby avoiding the possible species differences that occur in large and small animals.
In WP6 the porcine experiments met all requirements for animals according to EU legislation and national SJVFS 2012:26 (L 150). The animals were provided with food and water ad libitum. Animal caretakers and veterinary nurses handled the animals during a 2-week acclimatization period, and clinical examinations was performed daily by an experienced veterinarian. The 3Rs were thoroughly considered, e.g. epidural morphine was provided to minimize pain from abdominal surgery. At the end of the experiment, after 3 months, the pigs were euthanized in their home pens with an overdose of pentobarbital sodium.
The preclinical toxicology and pharmacokinetic studies of compstatin AMY-101 (WP8) in non-human primates (NHP) were performed by contract research organizations (CRO) and was performed according to the Principles of Good Laboratory Practice (GLP). The selected CROs had permission to conduct toxicity tests in NHP under GLP conditions. The studies were performed according to the EU 2010/63 Directive and all procedures were performed taking into consideration the principles of the 3Rs. Thus, the number of animals included was as low as possible and handling procedures were strictly performed according to animal welfare legislation. A national committee for animal experiments approved the applications before onset of the studies. The result from this preclinical studies supported the assumption that a coming phase I trial would be uncomplicated and with ethical concerns.
List of Websites:
Bo Nilsson, Professor
Uppsala Universitet
Department of Immunology, Genetics and Pathology
Clinical Immunology
Email: bo.nilsson@igp.uu.se
Mobile phone: +46 70 9423977
Fax: 018-553601
Visiting address:
Dag Hammarskjölds väg 20
751 85 Uppsala
Postal address:
Rudbecklaboratoriet
751 85 UPPSALA
www.kidneydirekt.org
www.kidneydirekt.com
