Final Report Summary - VASOPLUS (Placental Growth Factor (PlGF): new diagnostic and therapeutic applications in cardiovascular disease)
Ischemic heart disease and Peripheral arterial occlusive disease (PAOD) are the major cause of severe morbidity and mortality in Western societies: all situations with a restriction of blood flow and cardiac dysfunction or intermittent claudication in arms and legs as consequence. Initially, it was thought that delivery of proangiogenic factors would be a straightforward approach for ischemic disease therapy. However, many therapeutic candidates failed during the clinical development mainly due to high metabolic clearance (e.g. FGF) or the observation of side effects (e.g. VEGF). Placental growth factor (PIGF) belongs also the VEGF-related growth factor family but is an outstanding drug candidate since many preclinical proof-of concept studies have described its high angiogenic and arteriogenic efficiency in pathological situations without affecting the vasculature in normal tissues. The ultimate goal of VASOPLUS was the development of a stable PIGF formulation as a safe and efficacious medicine for cardiac disorders and arterial insufficiency. Additionally, this project evaluated the potential of PIGF as a biomarker of cardiovascular disease.
Six contractors were involved in this project:
- Geymonat (GT) initiated translational research (production and toxicology);
- Eurogentec (EGT) supported production process optimisation and industrial upscaling;
- Cardiology Department of the KULeuven (CARD) with a longstanding expertise in the area of cardiac function and regeneration of cardiac tissue;
- Consiglio Nazionale delle Ricerche (CNR), experienced in gene variability analysis and in-depth correlation studies with multifactoral diseases;
- Roche Diagnostics (RD), specialised in identification of biomarkers and translation into diagnostic products;
- ThromboGenics (TG), focussed on development of vascular biopharmaceuticals and coordinator of this project.
The present STREP encompassed the following main objectives:
1) research on PIGF-compound improvements and alternative isoforms, industrial production process optimisation of the selected lead candidate and finally, production upscaling;
2) 'translational research' to define the potential of recombinant PlGF-1 protein as safe and efficient therapeutic for limb and heart ischemic disorders in animal models;
3) development of a diagnostic test for measurement of systemic PlGF levels, determination of PIGF levels and other biomarkers in samples from a well- characterised Italian cohort and correlation with different cardiovascular diseases.
The industrial production development started with optimisation of a process thawas using transfected prokaryotic cells (E. coli) over-expressing recombinant human PIGF-1 isoform. The proteins were mainly secreted as unglycosylated dimers connected by one or two disulfide bonds but quality and stability control revealed that the starting material was not homogeneous and stable enough to meet the-criteria required for a biopharmaceutical drug. In depth characterisation indicated the presence of a free cystein residue as a source for polymerisation and instability.
GT constructed a significantly more stable mutated PlGF-1 variant by substitution of the free cystein that prevented protein aggregation in solution and in several kinds of carbopol-based gels and lotion containing alcohol, which is necessary for optimal treatment in settings as wound healing. Subsequently, EGT succeeded in developing a robust and reproducible production process yielding a homogeneous mutein that was characterised as a stable PlGF variant showing high activity in vitro.
As back-up strategy, EGT started with an alternative process development of a third generation of stabilised non-mutated PlGF-1. In addition, TG produced glycosylated derivates of PIGF-1 and PlGF-2 in order to investigate the role of glycosylation because PlGF is secreted naturally as a glycoprotein. In vitro activity tests demonstrated that the glycosylation process did not promote receptor recognition.
In order to determine the potential of PIGF as therapeutic agent for cardiac and peripheral limb ischemia, the present STREP comprised validation of two new relevant animal models. Functional biological PlGF-activity was preliminary tested in murine models of heart and limb ischemia but final proof-of-concept activity studies were performed in rabbit and pig models for limb and heart ischemic diseases respectively because of species specificity and model sensitivity. Local treatment with PlGF-1 wild-type induced stable collateral growth in the rabbit ischemic hind limb and a relevant improvement in limb perfusion seven days after blood flow-limiting lesion in the proximal arterial conduit. Delivery of mutein, however, restored the peripheral blood conductance less efficiently although a dose-related effect was observed on the arterial density. ln order to investigate therapeutic efficiency of PIGF on revascularisation in ischemic heart CARD established a new relevant model of porcine chronic myocardial ischemia in pigs a model closely related to the human situation. Systemic delivery of mutein produced significantly increased myocardial function and blood flow in and around the ischemic region. Unfortunately, this vascular improvement did not lead to a higher global heart perfusion capacity.
In conclusion, PIGF-1 delivery did not induce any side effects or signs of hypervascularisation but the preclinical objectives in both animal models did not reach the general endpoints needed to justify further product development of the most promising lead compound, PlGF-1 mutein. Besides translational research and production development of PlGF as medicine for pro-angiogenic treatments the present STREP evaluated the value of PIGF as a diagnostic test for angiogenic disorders. PIGF and VEGF levels as well as possible variants of their genes were correlated with the health status of human populations in order to define their role and the modulation of action in a particular pathogenesis. A database developed by CNR and holding the description of the health status, the genealogy and a genome wide scan from well-characterised genetically isolated populations in Southern italy, Campora, Gioi and Cardile made this huge and unique association study feasible. CNR and RD analysed PIGF, VEGF levels and other biomarkers (TNT-hs, NT-proBNP, CRP, sFIt-1) in 2 100 samples collected from the individuals comprised in the above described databank and investigated their association with traits or diseases as hypertension, type 2 diabetes, obesity and atherosclerosis. PIGF levels were measured using a fully automated one-step enzyme immunoassay based on electrochemiluminescence technology and specially developed by RD for diagnostic use. Elevated PIGF levels could be associated with obesity, diabetes, hypertension as well as atherosclerosis giving evidence for use as a biomarker for the diseases. Subsequently, CNR identified the genetic variants influencing the plasma level of PIGF traits and investigated the association of five common -PIGF and VEGF haplotypes with a trait or disease. No differences were found in the distribution of the VEGF haplotypes between healthy controls and cases but one PIGF haplotype that was also related to higher levels of PIGF was found significantly more frequent in diabetic patients. Thus, this specific PIGF-haplotype can be considered as a risk factor for diabetes.
ln summary, this STREP confirmed and highlighted the therapeutic potential of PIGF as safe and efficient medicine for peripheral and cardiovascular application in established and new preclinical ischemia models. An unglycosylated PIGF-1 wild-type formulation reached the preclinical endpoints but unfortunately did not meet the quality criteria required for a biopharmaceutical drug. ln parallel, the current project demonstrated a close relationship between PIGF blood levels and the incidence of cardiovascular disorders giving crucial evidence to consider PIGF as a valuable biomarker for common cardiovascular disorders. The development of a reliable and sensitive diagnostic test for PIGF determination accelerates the way to commercialisation for a first diagnostic use.
Six contractors were involved in this project:
- Geymonat (GT) initiated translational research (production and toxicology);
- Eurogentec (EGT) supported production process optimisation and industrial upscaling;
- Cardiology Department of the KULeuven (CARD) with a longstanding expertise in the area of cardiac function and regeneration of cardiac tissue;
- Consiglio Nazionale delle Ricerche (CNR), experienced in gene variability analysis and in-depth correlation studies with multifactoral diseases;
- Roche Diagnostics (RD), specialised in identification of biomarkers and translation into diagnostic products;
- ThromboGenics (TG), focussed on development of vascular biopharmaceuticals and coordinator of this project.
The present STREP encompassed the following main objectives:
1) research on PIGF-compound improvements and alternative isoforms, industrial production process optimisation of the selected lead candidate and finally, production upscaling;
2) 'translational research' to define the potential of recombinant PlGF-1 protein as safe and efficient therapeutic for limb and heart ischemic disorders in animal models;
3) development of a diagnostic test for measurement of systemic PlGF levels, determination of PIGF levels and other biomarkers in samples from a well- characterised Italian cohort and correlation with different cardiovascular diseases.
The industrial production development started with optimisation of a process thawas using transfected prokaryotic cells (E. coli) over-expressing recombinant human PIGF-1 isoform. The proteins were mainly secreted as unglycosylated dimers connected by one or two disulfide bonds but quality and stability control revealed that the starting material was not homogeneous and stable enough to meet the-criteria required for a biopharmaceutical drug. In depth characterisation indicated the presence of a free cystein residue as a source for polymerisation and instability.
GT constructed a significantly more stable mutated PlGF-1 variant by substitution of the free cystein that prevented protein aggregation in solution and in several kinds of carbopol-based gels and lotion containing alcohol, which is necessary for optimal treatment in settings as wound healing. Subsequently, EGT succeeded in developing a robust and reproducible production process yielding a homogeneous mutein that was characterised as a stable PlGF variant showing high activity in vitro.
As back-up strategy, EGT started with an alternative process development of a third generation of stabilised non-mutated PlGF-1. In addition, TG produced glycosylated derivates of PIGF-1 and PlGF-2 in order to investigate the role of glycosylation because PlGF is secreted naturally as a glycoprotein. In vitro activity tests demonstrated that the glycosylation process did not promote receptor recognition.
In order to determine the potential of PIGF as therapeutic agent for cardiac and peripheral limb ischemia, the present STREP comprised validation of two new relevant animal models. Functional biological PlGF-activity was preliminary tested in murine models of heart and limb ischemia but final proof-of-concept activity studies were performed in rabbit and pig models for limb and heart ischemic diseases respectively because of species specificity and model sensitivity. Local treatment with PlGF-1 wild-type induced stable collateral growth in the rabbit ischemic hind limb and a relevant improvement in limb perfusion seven days after blood flow-limiting lesion in the proximal arterial conduit. Delivery of mutein, however, restored the peripheral blood conductance less efficiently although a dose-related effect was observed on the arterial density. ln order to investigate therapeutic efficiency of PIGF on revascularisation in ischemic heart CARD established a new relevant model of porcine chronic myocardial ischemia in pigs a model closely related to the human situation. Systemic delivery of mutein produced significantly increased myocardial function and blood flow in and around the ischemic region. Unfortunately, this vascular improvement did not lead to a higher global heart perfusion capacity.
In conclusion, PIGF-1 delivery did not induce any side effects or signs of hypervascularisation but the preclinical objectives in both animal models did not reach the general endpoints needed to justify further product development of the most promising lead compound, PlGF-1 mutein. Besides translational research and production development of PlGF as medicine for pro-angiogenic treatments the present STREP evaluated the value of PIGF as a diagnostic test for angiogenic disorders. PIGF and VEGF levels as well as possible variants of their genes were correlated with the health status of human populations in order to define their role and the modulation of action in a particular pathogenesis. A database developed by CNR and holding the description of the health status, the genealogy and a genome wide scan from well-characterised genetically isolated populations in Southern italy, Campora, Gioi and Cardile made this huge and unique association study feasible. CNR and RD analysed PIGF, VEGF levels and other biomarkers (TNT-hs, NT-proBNP, CRP, sFIt-1) in 2 100 samples collected from the individuals comprised in the above described databank and investigated their association with traits or diseases as hypertension, type 2 diabetes, obesity and atherosclerosis. PIGF levels were measured using a fully automated one-step enzyme immunoassay based on electrochemiluminescence technology and specially developed by RD for diagnostic use. Elevated PIGF levels could be associated with obesity, diabetes, hypertension as well as atherosclerosis giving evidence for use as a biomarker for the diseases. Subsequently, CNR identified the genetic variants influencing the plasma level of PIGF traits and investigated the association of five common -PIGF and VEGF haplotypes with a trait or disease. No differences were found in the distribution of the VEGF haplotypes between healthy controls and cases but one PIGF haplotype that was also related to higher levels of PIGF was found significantly more frequent in diabetic patients. Thus, this specific PIGF-haplotype can be considered as a risk factor for diabetes.
ln summary, this STREP confirmed and highlighted the therapeutic potential of PIGF as safe and efficient medicine for peripheral and cardiovascular application in established and new preclinical ischemia models. An unglycosylated PIGF-1 wild-type formulation reached the preclinical endpoints but unfortunately did not meet the quality criteria required for a biopharmaceutical drug. ln parallel, the current project demonstrated a close relationship between PIGF blood levels and the incidence of cardiovascular disorders giving crucial evidence to consider PIGF as a valuable biomarker for common cardiovascular disorders. The development of a reliable and sensitive diagnostic test for PIGF determination accelerates the way to commercialisation for a first diagnostic use.