Patent Application
20230119290
The present invention relates to a column comprising a vascular endothelial growth factor (VEGF) dimer molecule, a method for preparing such a column, a VEGF dimer molecule, an expression vector and a recombinant host cell encoding for a VEGF dimer, as well as uses and methods related thereto.
The vascular endothelial growth factor (VEGF) family is a sub-family of growth factors, which is termed the platelet-derived growth factor family of cystine-knot growth factors. Members of the VEGF family are VEGF-A (often referred to as VEGF), VEGF-B, VEGF-C, VEGF-D and Placental growth factor (PIGF). VEGF family members usually function as ligands interacting with at least one of the cellular VEGF receptor tyrosine kinases VEGFR-1, VEGFR-2 and VEGFR-3. Thereby they may operate as important signaling proteins involved in the formation of blood vessels, such as vasculogenesis (blood vessel formation in the embryo by de novo formation of the embryonic circulatory system) and angiogenesis (the formation of blood vessels from pre-existing vessels). Defects in the level of interaction of VEGF family members and their VEGF receptors may lead to severe diseases, such as preeclampsia in pregnant women.
Preeclampsia is a potentially life-threatening multisystem disease affecting 4-8% of pregnant women after the 20th week of gestation (Sibai, 1998; Redman, 2005; Walker 2000). In detail, preeclampsia is usually defined as new-onset hypertension and damage to another organ system, most commonly the kidney indicated by loss of protein in the urine (proteinuria). The condition may progress to life-threatening generalized endothelial dysfunction and thrombotic microangiopathy manifesting with seizures, stroke or multiorgan failure, also called eclampsia or HELLP syndrome (Hemolysis Elevated Liver enzymes Low Platelets).
The pathophysiologic mechanism of preeclampsia is an imbalance of angiogenic and anti-angiogenic factors in the maternal circulation. According to current knowledge, placental ischemia leads to increased expression and systemic release of sFlt-1, the soluble splice variant of VEGF receptor 1 (VEGFR-1), most likely from trophoblast cells (Maynard et al., 2003). sFlt-1 acts as a potent VEGF and placental growth factor (PIGF) antagonist by scavenging and neutralizing these molecules. As a result, the endothelium is deprived of its constant trophic signal, leading to generalized endothelial dysfunction with hypertension and proteinuria being the first hallmark symptoms of disease and, consequently, severe consequences for mother and consequently also her fetus.
In patients with preeclampsia, increased sFlt-1 and decreased circulating PIGF plasma levels are increasingly evolving as powerful predictive markers of imminent disease and can be detected up to five weeks before the onset of symptoms (Levine et al., 2004).
So far, there are only small feasibility pilot studies in clinical trial phase 2 on extracorporeal apheresis (dextran sulfate, H.E.L.P.) and also plasma separation. Effective therapeutic and prophylactic measures for severe preeclampsia are lacking to date except for pre-mature termination of pregnancy (ACOG, 2019). This is especially problematic in patients with early preeclampsia before the 34th week estimated gestational age (EGA), when prolongation of pregnancy may be beneficial to the fetus but at the same time detrimental to the mother resulting in an unresolvable therapeutic dilemma.
Therapeutic approaches are e.g., the systemic administration of recombinant proteins, such as VEGF121 or PIGF. This resulted in attenuation of symptoms of preeclampsia in animal models (Makris et al., 2016; Li et al., 2007). However, the administration of VEGF and PIGF is limited by serious dose limiting side effects in the mother in case of VEGF and the short half-life limiting the route of PIGF (Eddy et al., 2018). In addition, the administration of stabilized siRNA reduced placental overexpression of sFlt-1 without affecting Flt-1 mRNA in mouse and baboon preeclampsia models (Turanov et al., 2018).
For further development of therapies, understanding the complex pathophysiology of preeclampsia is essential. According to mouse studies and in-situ hybridization experiments in human placenta, trophoblast-derived sFlt-1 protects the fetus from maternal VEGF, which is released in response to placental hypoperfusion and ischemia (Fan et al., 2014). According to this model overflow of placental sFlt-1 into the maternal circulation represents an unintended excess of a primarily physiologic response. Thus, therapeutic approaches targeting the expression of sFlt-1 require caution since they might compromise the physiologic function of sFlt-1 in the placenta and expose the fetus to increased levels of maternal VEGF (Turanov et al., 2018). Circulating sFlt-1 in the maternal blood stream contributes only little to the overall sFlt-1 burden, which mainly accumulates in the extracellular matrix due to its high positive charge. However, only the smaller circulating proportion of sFlt-1 seems to cause endothelial dysfunction and multisystemic disease. Bearing in mind the protective role of sFlt-1 in the placenta and the detrimental function of circulating sFlt-1 the principle of extracorporeal removal of sFlt-1 is captivating.
Therefore, besides the systemic administration of recombinant proteins, extracorporeal strategies are examined to remove sFlt-1 in human pregnancy to restore angiogenic balance in preeclamptic pregnancies. Extracorporeal apheresis of sFlt-1 primarily targets circulating sFlt-1 and only secondarily affects tissue and placental levels of sFlt-1 after redistribution.
One approach is the extracorporeal adsorption of circulating sFlt-1 by ionexchange chromatography by employing dextran sulfate in apheresis of pregnant women with severe early preeclampsia (Thadhani et al., 2011; Thadhani et al., 2016). In this approach, positively charged sFlt-1 is unspecifically retained by the negatively charged dextran sulfate matrix. However, charge dependent ligands, such as dextran sulfate, are very unspecific and may, therefore, also lead to the unwanted removal of other proteins.
Further approaches of extracorporeal adsorption of circulating sFlt-1 are antibody based approaches to reduce sFlt-1 (e.g. US 2010/0247650 A1). However, complexed VEGF and PIGF are also eliminated concomitantly and reduce efficacy of the intervention with regard to restoring the angiogenic balance. Furthermore, the immunogenic potential of sFlt-1 specific antibodies as well as adverse effects due to leakage of antibody from the column are uncertain.
In another approach, ligands, such as PIGF, are immobilized on a matrix and applied in apheresis of pregnant women suffering from preeclampsia (e.g. US 2010/0247650 A1).Thereby, PIGF columns remove sFlt-1 by binding to its receptor site. However, due to the low affinity of PIGF, the capturing PIGF-molecule is not able to compete with complexed VEGF. Therefore PIGF-columns only remove free sFlt-1 or sFlt-1 from circulating PIGF-sFlt-1 complexes. PIGF columns do not remove VEGF-complexed sFlt-1 and do not liberate VEGF. Therefore, this approach only partly removes sFlt-1 and, in comparison to potential competitive apheresis systems, does not restore the angiogenic balance.
A further approach applies magnetic beads functionalized with monomeric truncated VEGF in apheresis to capture sFlt-1 (Trapiella-Alfonso et al., 2019). This approach used a bacterial expressed VEGF truncation (VEGF95) and biotinylated beads. However, this approach is not feasible for application in humans due to non-covalent linkage of the ligand to the matrix and unacceptable immunogenity of the avidin-biotin-based interaction (Jain and Cheng, 2017). In addition, the application of bacterial expressed VEGF95 has additional limitations. Although glycosylation of VEGF was reported to not affect VEGF binding affinity and activity (Claffey et al., 1995; Peretz et al., 1992), post translational modification specific to bacteria may aggravate immunogenicity (Kuriakose et al., 2016). In addition, the truncated VEGF isoform VEGF95 only displays reduced interaction with sFlt-1 due to lacking residues in the receptor binding interface. Also this approach does not lead to the removal of VEGF165-complexed sFlt-1 and does not cause the liberation of VEGF165.
There is a need to provide means for the treatment of preeclampsia. Especially, there is a need for providing means that allow the efficient removal of sFlt-1 from the blood of patients so that preeclampsia can be efficiently treated in humans.
A column comprising a vascular endothelial growth factor (VEGF) dimer molecule comprising a first and a second VEGF molecule is provided.
The invention further relates to a VEGF dimer molecule comprising a first and a second VEGF molecule, wherein the first and/or the second VEGF molecule have a length of 122 to 250 amino acids.
Moreover, the invention relates to a method for preparing a column of the invention comprising the steps of:
Further, a VEGF dimer molecule of the invention for use in the treatment of preeclampsia characterized in that the VEGF dimer molecule is bound to a column for apheresis is provided.
The invention further relates to an expression vector comprising a nucleic acid sequence encoding the VEGF dimer molecule of the invention.
Moreover, the invention relates to a recombinant host cell line comprising the VEGF dimer molecule of the present invention, comprising the expression vector of the present invention and/or comprising a nucleic acid sequence encoding the VEGF dimer molecule of the present invention.
A method for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood comprising incubating the column or the VEGF dimer molecule of the invention with the blood and separating sFlt-1 from the blood and/or releasing VEGF from complexes with sFlt-1 into the blood is also provided.
In addition, the invention relates to the use of the column or the VEGF dimer molecule of the invention for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood.
In a first aspect, the invention relates to a column comprising a vascular endothelial growth factor (VEGF) dimer molecule comprising a first and a second VEGF molecule.
Surprisingly, the results of the examples show that sFlt-1 adsorption from human serum was significantly higher for a column comprising a dimer of truncated VEGF of the present invention called scVEGF165 in comparison to monomeric PIGF (moPIGF) or monomeric (moVEGF165) when coupled to all resins tested. Especially, according to the example, the largest reduction of 86.16 % sFlt-1 in a single run was achieved with columns, where said dimer of the invention (scVEGF165) was immobilized on an agarose matrix (
In general, it can be concluded from the results of the examples shown below that the unique characteristic of an apheresis with a VEGF dimer molecule of the invention compared to antibody-mediated apheresis and monomeric VEGF- or PIGF-based approaches is the enhanced affinity for sFlt-1 and thus the capability to efficiently release VEGF and PIGF (
To sum up, the invention describes for the first time a specific VEGF-based sFlt-1 apheresis system to treat preeclampsia that is applicable in vivo. By exploiting the unique molecular characteristics of the VEGF molecule, it was possible to generate highly specific apheresis columns to immobilize sFlt-1 and at the same time competitive release complexed VEGF and PIGF to restore the angiogenic balance in patients with preeclampsia. Due to the complex and highly structured morphology of VEGF dimers exposing the receptor binding site, use of full-length VEGF dimers as compared to shorter peptide sequences yield better affinity.
Columns for performing apheresis are known in the art (Thadhani et al., 2011; Thadhani et al., 2016). The term “column” according to the present invention describes any device comprising a lumen with two open ends. For example, this means that in case of a column for apheresis, blood from the patient is led into the lumen of the column via the first open end, and, after passing the lumen, is led out of the lumen via the second open end.
The column of the present invention usually is capable of carrying the matrix for a chromatographic separation method. Preferably, the column of the invention is suitable for separating components of a mixture, such as blood or blood plasma. For example, the column may be a chromatography column, such as used for affinity chromatography.
The column may further be made of any material known to be suitable for a chromatography column by the person skilled in the art, such as metal, glass and/or plastic (e.g. polycarbonate). In general, the material is suitable for medical applications, such as apheresis. Moreover, the column may have any size or volume. Preferably, the column has a volume suitable to be applicable in apheresis (e.g. from 20 to 300 ml). Moreover, the column of the invention may have any shape known to the person skilled in the art to be suitable for chromatography methods, such as a cylindrical shape or a tube. Usually, the cavity of the column according to the present invention is packed with the matrix.
The column may be suitable for being applied in medical methods, such as in extracorporeal therapy, e.g. apheresis, whereby the blood or blood plasma of a person may be passed through the column leading to the separation of one or more particular constituent and returning the remaining one or more constituents to the circulation. Moreover, the column may be sterilized by e.g. irradiation, such as gamma-ray irradiation, electron beam irradiation or x-ray irradiation.
The column of the present invention comprises a vascular endothelial growth factor (VEGF) dimer molecule comprising a first and a second VEGF molecule.
VEGF molecules are known in the art. The VEGF molecule comprised according to the present invention in the VEGF dimer molecule may be any vascular endothelial growth factor protein molecule known to the person skilled in the art including truncated forms thereof and may be from any species. For example, the VEGF molecule may be mammalian VEGF, such as mouse, rat or human VEGF. Preferably, the VEGF molecule is human VEGF. Preferably the VEGF molecule is VEGF-A, VEGF-B, VEGF-C, VEGF-D or PIGF, more preferably the VEGF molecule is VEGF-A, VEGF-B or PIGF, even more preferably the VEGF molecule is VEGF-A or VEGF-B and most preferably the VEGF molecule is VEGF-A.
Preferably the VEGF molecule is human VEGF-A, human VEGF-B, human VEGF-C, human VEGF-D or human PIGF, more preferably the VEGF molecule is human VEGF-A, human VEGF-B or human PIGF, even more preferably the VEGF molecule is human VEGF-A or human VEGF-B and most preferably the VEGF molecule is human VEGF-A.
The VEGF molecule comprised in the VEGF dimer of the column of the first aspect of the invention may be of any length. The VEGF molecule comprised in the VEGF dimer of the column of the first aspect of the invention may be full-length VEGF, preferably full-length VEGF-A. For example, the VEGF molecule may comprise or consist of the sequence of human full-length VEGF-A (SEQ ID NO: 1).
Moreover, the VEGF molecule may be a truncated form of VEGF, such as a truncated form of VEGF-A. Preferably the VEGF molecule of the column of the first aspect of the invention may be a truncated form of human VEGF, more preferably a truncated form of human VEGF-A, such as human VEGF-A121, human VEGF-A138, human VEGF-A145, human VEGF-A162, human VEGF-A165, human VEGF-A165b, human VEGF-A189 or human VEGF-A206 (whereby the numbers correspond to the numbers of proteins in the respective protein based on the natural human VEGF-A sequence).
In an especially preferred embodiment, the VEGF molecule is human VEGF-A165.
The truncated VEGF molecule may only comprise a truncated natural VEGF molecule sequence or additional extensions as describe below. For example, the first and/or the second VEGF molecule may be truncated down to a length of 122 amino acids, preferably 165 amino acids.
Preferably, the first VEGF molecule comprised in the VEGF dimer of the column of the first aspect of the invention comprises the N-terminal helix (24-35), the loop connecting β3 to β4 (69-74) and strand β7 (111-114) and the second VEGF molecule comprises residues from strand β2 (54-56) and from strands β5 and β6 together with the connecting turn (79- 91). In that case, the first and second VEGF molecule may dimerize in anti-parallel orientation and, thereby, together comprise the region responsible for sFlt-1 binding when forming the VEGF dimer molecule, i.e. sFlt-1 the binding site may be composed of respective residues from both monomers.
It is also possible in case of a VEGF dimer molecule as used in the column of the first aspect of the invention that the truncated first VEGF molecule consists of amino acids 1 to 114, i.e. comprising the sFlt-1 binding site comprising the N-terminal helix (24-35), the loop connecting β3 to β4 (69-74) and strand β7 (111-114), and the truncated second VEGF monomer consists of amino acids 1 to 91, comprising the residues from strand β2 (54-56) and from strands β5 and β6 together with the connecting turn (79- 91).
Also preferably, the first and/or second VEGF molecule comprise the VEGF amino acid residues Asp63, Glu64 and Glu67 (numbers correspond to those of the natural occurring human VEGF-A amino acid sequence).
Moreover, the amino acid sequence of the VEGF molecule may be a natural occurring sequence or may be mutated. The VEGF molecule may comprise no, one, two, three, four or more mutations. Mutations may affect any amino acid of a VEGF molecule. For example, amino acids may be mutated to enhance binding affinity of a VEGF molecule to sFlt-1. Mutations may also comprise the exchange of one or more amino acids for amino acids having the same or similar charge and/or size.
The VEGF molecule may comprise any form of posttranslational modification known to the person skilled in the art, such as phosphorylation, glycosylation and/or acetylation. Moreover, the VEGF molecule may comprise one, two, three, four or more intramolecular disulfide bonds.
Further, in the column of the present invention, the first and the second VEGF molecule are part of a vascular endothelial growth factor (VEGF) dimer molecule.
The term “VEGF dimer molecule” according to the present invention describes any molecule comprising at least two VEGF molecules, preferably two VEGF molecules.The at least two VEGF molecules that are part of the VEGF dimer molecule may interact via one, two, three, four or more amino acids by ionic binding, covalent binding, van der Waals forces, hydrogen bridge bonds, hydrophobic interactions and/or electrostatic interaction.
Preferably, the at least two VEGF molecules that are part of the VEGF dimer molecule may be connected by at least one covalent bond. More, preferably the at least two VEGF molecules that are part of the VEGF dimer molecule may be connected by one covalent bond, e.g. forming a single-chain molecule. For example, in case of a first and a second VEGF molecule, the nucleic acid sequences encoding of both VEGF molecules may be connected to one nucleic acid sequence molecule leading to the expression of one amino acid molecule comprising both VEGF molecules.
The first and the second VEGF molecule in a VEGF dimer molecule may further be linked by one, two, three, four or more intermolecular and/or intramolecular disulfide bonds. For example, the VEGF dimer molecule may form an open structure of two VEGF molecules (such as scVEGF165 molecules) linked by one or more intramolecular disulfide bonds.
Alternatively, the first and the second VEGF molecule in a VEGF molecule dimer may not be linked by any intramolecular disulfide bond.
Any amino acid involved in one of above-mentioned intramolecular or intermolecular disulfide bridges or being directly or sterically adjacent may be mutated.
Dimerization of the first and the second VEGF molecule to form a VEGF dimer molecule may occur in an antiparallel side-by-side fashion with intermolecular disulfide bonds between the single monomers generating a planar surface for the interaction with the receptor protein (Flt-1/sFlt-1). The Flt-1 binding site may comprise residues of 3 beta sheets (β3; β4; β7) as well as the N-terminal helix of one monomer and residues from 3 complementary beta sheets (β2; β5; β6) from the other. Mutational strategies to enhance VEGF-affinity for sFlt-1 may affect any one of these residues or none of them at all.
Moreover, also steric effects may affect sFlt-1 affinity in multimeric forms of VEGF and PIGF and, therefore, may also be altered by one or more mutations.
Moreover, the VEGF dimer molecule may further be part of a VEGF multimer molecule, such as a VEGF trimer, a VEGF tetramer or any form of a VEGF multimer comprising any number of VEGF molecules. Therefore, the column may also comprise a VEGF trimer, a VEGF tetramer or any form of a VEGF multimer. Further, the column may comprise VEGF molecules of one multimer type only, such as only VEGF dimers, only VEGF trimers or only VEGF tetramers, or the column may comprise any mixture of multimers, such as a mixture of VEGF dimers, VEGF trimers, VEGF tetramers and/or VEGF multimers comprising any number of VEGF molecules.
Moreover, the VEGF dimer molecule may be part of a VEGF multimeric structure. For example, a first VEGF dimer molecule may interact (e.g. via intermolecular disulfide bonds) with a second VEGF dimer molecule allowing the formation of tetramers, or multimeric chains of VEGF molecules.
In an especially preferred embodiment, the VEGF dimer molecule of the column of the invention is the VEGF dimer molecule according to the second aspect of the invention discussed below.
In a second aspect, the invention relates to a VEGF dimer molecule comprising a first and a second VEGF molecule, wherein the first and/or the second VEGF molecule have a length of 122 to 250 amino acids.
Except for the length of the first and/or second VEGF molecule, which in the second aspect of the invention is restricted to 122 to 250 amino acids, while there are no restrictions in VEGF molecule according to the first aspect of the invention, all embodiments and features described above with respect to the first and second VEGF molecule of the column according to the first aspect of the invention also apply to this second aspect. For example, if the first and/or second VEGF molecule are truncated, the VEGF molecule of the second aspect of the invention preferably may be a truncated form of human VEGF having a length of 122 to 250 amino acids, more preferably a truncated form of human VEGF-A having a length of 122 to 250 amino acids, such as, human VEGF-A138, human VEGF-A145, human VEGF-A162 , human VEGF-A165 , human VEGF-A165b , human VEGF-A189 or human VEGF-A206 (whereby the numbers correspond to the numbers of proteins in the respective protein based on the natural human VEGF-A sequence).
Further, with the exception of the restriction to the lengths of the first and/or second VEGF molecule, all embodiments and features described above with respect to the VEGF dimer molecule of the column according to the first aspect of the invention also apply to the second aspect.
The following embodiments further describe both, the column of the first aspect of the invention and the VEGF dimer molecule of the second aspect of the invention, if not indicated that they only concern the first or second aspect only.
The VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention can be chemically synthesized by conventional methods. Methods for chemically synthesizing the VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention are well known to the person skilled in the art.
Furthermore, the VEGF dimer molecule may be produced by recombinant protein expression, e.g. it may be expressed in eukaryotic cells, such as in human cells lines (e.g. HEK293T Epstein-Barr virus nuclear antigen (EBNA) cell lines), mouse cell lines, rat cell lines, hamster cell lines, yeast cells or insect cells. Suitable methods for recombinant protein expression of the VEGF dimer molecule are well known to the person skilled in the art. For example, uncloned, double-stranded linear DNA fragments containing a nucleotide sequence representing the VEGF dimer may be customized, e.g. by codon optimization, and produced. Usually, DNA fragments are amplified using primers and cloned into an expression vector (such as the sleeping beauty transposon expression vector) carrying a protein tag, such as an N-terminal strep tag. In general, stable cell lines are generated by transfecting the vectors into the cells using a transfection method, such as applying a transfection reagent or electroporation. Afterwards, the protein may be isolated from the cells by harvesting the supernatant or the whole culture (if the protein is not secreted by the cells).
Subsequently, the produced VEGF dimer molecule is usually isolated and purified. Methods for isolating and purifying the VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention are well known to the person skilled in the art. For example, the VEGF dimer molecule can be purified using a filter technology or chromatography technique, such as affinity chromatography, high performance liquid chromatography (HPLC) or ion exchange chromatography, gel filtration, or other known methods. For example, the VEGF dimer molecule may be linked to and expressed with a protein-tag, such as a Strep tag or a glutathione S-transferase tag (GST-tag), Histidine-tag (HIS-tag) which allows purification via an affinity chromatography column, such as StrepTactin (e.g. Strep-Tactin®XT (IBA Lifesci-ence, Göttingen, Germany)) or glutathione or nickle affinity chromatography. During affinity chromatography or afterwards, the protein-tag is usually cut off from the VEGF dimer molecule by a protease. For example, the Strep tag may be cut off from the VEGF dimer molecule using thrombin protease.
The VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention may be synthesized or expressed as one molecule. Alternatively, The VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention may be synthesized or expressed in at least two parts and subsequently be connected. For example, the first and the second VEGF molecule of the VEGF dimer of the column of the first aspect of the invention or of the VEGF dimer of the second aspect of the invention may be synthesized or expressed separately and subsequently be connected.
Moreover, the VEGF dimer molecule or the first and/or the second VEGF molecule may be chemically modified, e.g. by adding chemical groups, such as posttranslational modifications. Suitable chemical modifications as well as methods for chemical modification are well known to the person skilled in the art, such as phosphorylation, glycosylation and/or acetylation.
In a preferred embodiment, the VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention is expressed by a eukaryotic cell.
The first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may be different. For example, they may differ in their length, in their amino acid sequence and/or in their posttranslational modifications. Alternatively, the first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may be identical.
In a further preferred embodiment, the first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention are identical.
The first and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may lack a part of or the whole VEGF N-terminal signal peptide MNFLLSWVHWSLALLLYLHHAKWSQA (SEQ ID NO: 2).
The part or the whole VEGF N-terminal signal sequence may be cleaved from the VEGF dimer sequence or the first and/or second VEGF molecule sequence before they are introduced in an expression vector or in the expression vector before introducing it into cells for recombinant expression. Moreover, the part or the whole VEGF N-terminal signal peptide may be cleaved from the synthesized or expressed first and/or second VEGF molecule before connecting them to a dimer or may be cleaved from the synthesized or expressed second VEGF molecule after connecting it to the first VEGF molecule to form a dimer. Methods for cutting of the N-terminal signal sequence from the VEGF nucleic acid or amino acid sequence are well known to the person skilled in the art as well as suitable nucleases or proteases.
In another preferred embodiment, the first and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention lack the N-terminal signal peptide.
Moreover, the first and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may be a full-length or truncated VEGF molecule extended by additional amino acids. In detail, the first and/or second VEGF molecule may be extended up to a length of 250 amino acids on their N- and/or C-terminus. Preferably, the first VEGF molecule is extended on its N-terminus and/or the second VEGF monomer is extended on its C-terminus. This may have the advantage that a possible misfolding of the first and the second VEGF molecule is prevented. For example, the first and/or the second VEGF molecule may be extended by a protein tag, such as a GST-tag, a HIS-tag, a Strep-tag or by addition of any sequence of amino acid residues. Moreover, the first and/or second VEGF molecule may still comprise remnants originally required for their production, such as restriction sites.
The first and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may have a length of 122 to 250 amino acids, preferably 125 to 225 amino acids, more preferably 140 to 200 amino acids, even more preferably 150 to 180 amino acids, especially more preferably 160 to 170 amino acids and most preferably 165 amino acids..
In a preferred embodiment, the first and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention have a length of 122 to 250 amino acids, preferably 125 to 225 amino acids, more preferably 140 to 200 amino acids, even more preferably 150 to 180 amino acids, especially more preferably 160 to 170 amino acids and most preferably 165 amino acids.
Moreover, the first VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity in comparison to SEQ ID NO: 3 and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity in comparison to SEQ ID NO: 4.
The term “at least 50% amino acid sequence identity” as used herein means that the amino acid sequence of the first and/or second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention has an amino acid sequence characterized in that, within a stretch of 100 amino acids, at least 50 amino acid residues are identical to the sequence of the corresponding sequence, e.g. of SEQ ID No: 3 and/or SEQ ID No: 4, respectively.
Sequence identity according to the present disclosure invention can, e.g., be determined by methods of sequence alignment in form of sequence comparison. Methods of sequence alignment are well known in the art and include various programs and alignment algorithms. Moreover, the NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Percentage of identity of mutants according to the present disclosure invention relative to the amino acid sequence of e.g. SEQ ID NO: 3 or SEQ ID NO: 4 is typically characterized using the NCBI Blast blastp with standard settings. The comparison of sequences and determination of percent identity between two amino acid sequences can also be accomplished with the program “BLAST 2 SEQUENCES (blastp)” (Tatusova et al., 1999) with the following parameters: Matrix BLOSUM62; Open gap 11 and extension gap 1 penalties; gap x_dropoff50; expect 10.0 word size 3; Filter: none.
Alternatively, sequence identity may be determined using the software GENEious with standard settings. Alignment results can be, e.g., derived from the Software Geneious (version R8), using the global alignment protocol with free end gaps as alignment type, and Blosum62 as a cost matrix.
Preferably, the first VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention has the amino acid sequence of SEQ ID NO: 3 and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention has the amino acid sequence of SEQ ID NO: 4.
In a further preferred embodiment, the first VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention has at least 50% amino acid sequence identity in comparison to SEQ ID NO: 3, preferably at least 60% amino acid sequence identity in comparison to SEQ ID NO: 3, more preferably at least 80% amino acid sequence identity in comparison to SEQ ID NO: 3 and most preferably at least 90% amino acid sequence identity in comparison to SEQ ID NO: 3 and/or the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention has at least 50% amino acid sequence identity in comparison to SEQ ID NO: 4, preferably at least 60% amino acid sequence identity in comparison to SEQ ID NO: 4, more preferably at least 80% amino acid sequence identity in comparison to SEQ ID NO: 4 and most preferably at least 90% amino acid sequence identity in comparison to SEQ ID NO: 4.
The first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may further be linked by a linker.
Alternatively, the VEGF dimer molecule may not comprise any linker. For example, the first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention may not be linked by a linker.
The term “linker” according to the present invention describes any spacer suitable to connect the first and the second VEGF molecule.
The linker may have a minimum length sufficient to allow flexibility of the first and/or second VEGF dimer relative to each other, e.g. leading to intermolecular multimerization of scVEGF, e.g leading to formation of scVEGF multimers, such as dimers and tetramers. The linker may have a length suitable to hamper intramolecular dimerization of VEGF-sequences.
The linker may comprise any material known to the person skilled in the art to be suitable for linking two protein molecules. For example, the linker may comprise any polymer of organic compounds known to the person skilled in the art, such as amino acids, nucleic acids, polysaccharides, polyethylene glycol and/or partial crosslinking, such as N-Hydroxysuccinimide (NHS). Preferably, the linker comprises amino acids and more preferably, the linker consists of amino acids. Moreover, the linker may be flexible or rigid.
For example, the nucleic acid sequences of the first and the second VEGF molecule may already be linked via the nucleic acid encoding the amino acid sequence of the linker and the whole construct may be expressed together as one VEGF dimer molecule (such as SEQ ID NO: 5) as described above.
The VEGF dimer molecule of the column according to the first aspect of the invention or according to the second aspect of the invention may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity in comparison to SEQ ID NO: 5. Preferably, the VEGF dimer molecule of the column according to the first aspect of the invention or according to the second aspect of the invention has 100% amino acid sequence identity in comparison to SEQ ID NO: 5.
Usually, the first and the second VEGF molecule may be linked by the linker, whereby the first end of the linker is connected to the C-terminal amino acid of the first VEGF molecule and the second end of the linker is connected to the N-terminal amino acid of the second VEGF molecule. Moreover, the first and the second VEGF molecule may be linked by the linker, whereby the first and/or second end of the linker are connected to an amino acid of the first and/or second VEGF molecule sequence that is not N-terminal or C-terminal.
In preferred embodiment, the first and the second VEGF molecule of the column according to the first aspect of the invention or of the VEGF dimer molecule according to the second aspect of the invention are linked by a linker.
If the linker comprises amino acids, the linker preferably has a length of 10 to 30 amino acids, preferably a length of 11 to 25 amino acids, more preferably a length of 12 to 20 amino acids, especially more preferably a length of 13 to 17 amino acids and most preferably a length of 14 amino acids
Moreover, the linker may comprise the sequence of SEQ ID NO: 6 (GSTSGSGKSSEGKG) or have a sequence identity of at least 50% amino acid sequence identity in comparison to SEQ ID NO: 6, preferably at least 70% amino acid sequence identity in comparison to SEQ ID NO: 6, more preferably at least 85% amino acid sequence identity in comparison to SEQ ID NO: 6 and most preferably at least 90% amino acid sequence identity in comparison to SEQ ID NO: 6. For example, it is possible to exchange single amino acids of SEQ ID NO: 6 for amino acids having the same or similar charge and/or size. Preferably, the linker comprises the sequence of SEQ ID NO: 6 and more preferably the linker consists of the sequence of SEQ ID NO: 6. Methods for determining the sequence identity of the linker in comparison to SEQ ID NO: 6 are well known to the person skilled in the art and comparable to those described above for determining sequence identity of the first and the second VEGF molecules to SEQ ID NOs: 3 and 4, respectively.
In a more preferred embodiment, the linker has a length of 10 to 30 amino acids, preferably a length of 11 to 25 amino acids, more preferably a length of 12 to 20 amino acids, especially more preferably a length of 13 to 17 amino acids and most preferably a length of 14 amino acids.
The VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention may be immobilized by a covalent bond to a matrix.
The matrix may be any molecule known to the person skilled in the art to be suitable for affinity chromatography of proteins. Usually, the matrix comprises chemical inert molecules. Especially, the molecules of the matrix may be suitable for clinical applications, such as apheresis. For example, they may have a low immunogenicity when applied in vivo.
Moreover, the molecules of the matrix generally allow covalent binding of the ligand VEGF dimer molecule. Therefore, a tag, such as a protein tag, may be connected to the VEGF dimer molecule and a matrix may be used that covalently binds this tag. Examples of suitable molecules of the matrix are well known to the person skilled in the art, such as sepharose, agarose and/or glutathione. Preferably, the matrix is sepharose or agarose, more preferably agarose. The matrix may further comprise a mixture of different molecules.
In another preferred embodiment, the VEGF dimer molecule of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention is immobilized by a covalent bond to a matrix.
In a third aspect, the invention relates to a method for preparing a column according to the first aspect comprising the steps of:
Regarding the features of steps a) and b), all embodiments and features described above with respect to the column of the first aspect of the invention and the VEGF dimer molecule according to the second aspect of the invention also apply to this the third aspect.
Methods for preparing a VEGF dimer molecule by eukaryotic expression are well known to the person skilled in the art and further described above. In particular, also the first and the second VEGF molecule and the linker are described above.
Further, in step b) of the method for preparing a column according to the third aspect of the invention the VEGF dimer molecule is immobilized to the matrix by a covalent bond. Features concerning the matrix are also described above in the context of a preferred embodiment of the column of the first aspect of the invention and are applicable to the third aspect of the invention.
The term “immobilizing the dimer of step a) on a matrix” according to the present invention describes the fixation of a VEGF dimer molecule to the matrix. Methods for immobilizing a dimer on a matrix are well known to the person skilled in the art. For example, the VEGF dimer molecule may be immobilized on a matrix when the matrix is already loaded onto a column or before loading it onto a column. In the first example, the matrix is first loaded onto the column. The VEGF dimer molecule may then be immobilized on that matrix by providing a solution comprising a VEGF dimer molecule and applying it to the matrix in the column. Due to specific interactions between the VEGF dimer molecule and the matrix, the VEGF dimer molecule usually stays in close proximity to the matrix or may bind to it, while the remaining substituents of the solution flow through the matrix and/or are removed by subsequent washing steps. Alternatively, in the second example, the matrix may be brought in contact with the VEGF dimer molecule leading to specific interactions between both of them. Subsequently, the matrix-VEGF dimer construct may be washed leading to removal of unspecifically binding components, but not of the specifically binding VEGF dimer molecules. Afterwards, the matrix with the immobilized VEGF dimer molecule may be loaded onto the column.
Moreover, in step b) the VEGF dimer molecule may be immobilized to the matrix by a covalent bond. Details of possible matrices that allow covalent binding of the VEGF dimer molecule are well known to the person skilled in the art and also described above for the column according to the first aspect of the invention.
The invention further relates to a column obtainable by the method according to the third aspect of the invention.
In a preferred embodiment, in step b) of the method for preparing a column according to the third aspect of the invention the VEGF dimer molecule is immobilized to the matrix by a covalent bond.
In a fourth aspect, the invention relates to the VEGF dimer molecule according to the second aspect for use in the treatment of preeclampsia characterized in that the VEGF dimer molecule is bound to a column for apheresis.
All embodiments and features described above with respect to the VEGF dimer molecule and the column as described in the first and/or second aspect of the invention also apply to this fourth aspect.
Moreover, the term “treatment of preeclampsia” according to the present invention comprises the treatment of all types of preeclampsia and all phases of this condition. For example, pregnant women may be treated as well as women, who recently gave birth and developed preeclampsia afterwards. Moreover, the treatment of preeclampsia also comprises the prophylactic treatment of pregnant women having a risk of developing preeclampsia, such as pregnant women suffering from antiphospholipid syndrome and/or hypertension or women having a sFlt-⅟PIGF ratio, which is higher than the average sFlt-⅟PIGF ratio in blood or serum in pregnant women, without showing symptoms of preeclampsia.
The term “apheresis” according to the present invention describes any extracorporeal medical therapy in which the blood or blood plasma of a person is passed through an apparatus that separates out one or more specific components, such as at least sFlt-1, and returns the remainder to the circulation. Thereby, beside the separation of sFlt-1, constituents bound to sFlt-1, such as VEGF and PIGF may be released into the blood and return to the circulation.
In general, apheresis methods and the required parameters are well known to the person skilled in the art. For example, an apheresis first comprises placing of a blood access. Therefore, needles are placed in peripheral veins of a patient. Single or double lumen catheters may also be used, for example in the internal jugular, subclavian of femoral veins. Usually, blood is then led from this first blood access into tubes or capillaries and pumped by a pump to an apheresis column, where sFlt-1 is separated from the blood and VEGF and PIGF are simultaneously released into the blood. The remaining constituents of the blood are then pumped by a second pump via further tubes or capillaries to a second blood access and returned to the patient’s blood circulation. Moreover, before being introduced into the apheresis column, the blood may be separated and only the blood plasma may be applied to the apheresis column. Subsequently, the cleaned blood plasma may be combined with the blood cells that were separated previously during preparation of blood plasma and both may be returned to the patient’s blood circulation.
The amount of blood plasma volume to be treated is usually the patient’s estimated plasma volume (EPV). EPV is usually calculated using the following formula:
Further, an apheresis usually comprises measures preventing, interrupting and terminating coagulation. These measures are well known to the person skilled in the art. For example, anticoagulants, such as citrate (e.g. sodium citrate or acid-citrate-dextrose (e.g. ACD-A)) or heparin may be added to the blood or blood plasma during apheresis. Suitable anticoagulants and the required dosage is well known to the person skilled in the art and may further depend on the clinical and physical condition of the patient.
One column may be used in one apheresis treatment or repeatedly for up to 5, 10, 15, 20 or 25 apheresis treatment applications.
In addition to the VEGF dimer molecule as described for the column according to the first aspect of the invention or of the second aspect of the invention, the apheresis column may also comprise further ligands that may interact with sFlt-1. Moreover, the apheresis column may comprise further ligands for removing blood components other than sFlt-1 during one apheresis application. For example, a plasma exchange (e.g. for removing a liquid portion of blood comprising harmful substances and replacing the plasma with a replacement solution) may be performed or low density lipoprotein (LDL) may be removed.
Moreover, the present invention relates to a method of using a column comprising the VEGF dimer molecule according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention to treat preeclampsia.
The invention also relates to a method for treating preeclampsia, comprising performing an apheresis using a column according to the first aspect of the invention or a VEGF dimer molecule according to the second aspect of the invention. All embodiments disclosed above also apply to these methods of the invention.
In a fifth aspect, the invention further relates to an expression vector comprising a nucleic acid sequence encoding the VEGF dimer molecule of the invention.
Suitable expression vectors are well known to the person skilled in the art. Preferably, the expression vector is suitable for the recombinant expression of a protein in eukaryotic cells.
For example, the expression vector may be a mammal-derived expression vector (e.g., pcDNA3 (manufactured by Invitrogen Corp.), pEGF-BOS (Mizushima and Nagata, 1990), pEF, and pCDM8), insect cell-derived expression vector (e.g., “Bac-to-BAC baculovirus expression system” (manufactured by Gibco BRL/Life Technologies, Inc.) and pBacPAK8), plant-derived expression vector (e.g., pMH1 and pMH2) or a yeast-derived expression vector (e.g., “Pichia Expression Kit” (manufactured by Invitrogen Corp.), pNV11, and SP-Q01). Preferably, the expression vector is the sleeping beauty transposon expression vector.
For the purpose of expression in animal cells such as CHO cells, COS cells, or NIH3T3 cells, the expression vector usually comprises a promoter for intracellular expression. Suitable promoters for intracellular expression are well known to the person skilled in the art. For example, SV40 promoter (Mulligan et al., 1979), MMLV-LTR promoter, EF1α promoter (Mizushima and Nagata, 1990), or CMV promoter may be used.
Moreover, the expression vector may further comprise a marker gene. Suitable marker genes are well known to the person skilled in the art. For example, the marker gene may be a drug resistance gene that is identifiable by a drug, such as neomycin, G418, etc., for transformed cells. Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
All features describing the VEGF dimer molecule of the invention according to the second aspect of the invention also apply to the VEGF dimer molecule encoded by the nucleic acid sequence in the expression vector of the fifth aspect of the invention.
In a sixth aspect, the invention relates to a recombinant host cell line comprising the VEGF dimer molecule of the present invention, comprising the expression vector of the present invention and/or comprising a nucleic acid sequence encoding the VEGF dimer molecule of the present invention.
Suitable recombinant host cell lines are well known to the person skilled in the art. For example, the recombinant host cell line may be a eukaryotic cell line, such as a human cells line (e.g. HEK293T Epstein-Barr virus nuclear antigen (EBNA) cell line), a mouse cell line, a rat cell line, a hamster cell line, a yeast cell line or an insect cell line.
All features describing the VEGF dimer molecule of the invention according to the second aspect of the invention or describing the expression vector of the fifth aspect of the invention also apply to the VEGF dimer molecule or the expression vector of the recombinant host cell line of the sixth aspect of the invention.
The present invention further relates to a matrix or a bead comprising the VEGF dimer according to the second aspect of the invention.
All features described for the matrix of the column according to the first aspect of the invention or the matrix used in the third aspect of the invention also apply to the matrix according to the present invention.
The bead may be any bead known to the person skilled in the art to be suitable for affinity chromatography of proteins. Usually, the bead comprises chemical inert molecules. Especially, the bead may be suitable for clinical applications, such as apheresis. For example, the bead may have a low immunogenicity when applied in vivo.
The bead may further be any molecule known to be suitable to be used in a column according to the first aspect of the present invention or to bind to a matrix described or used in the present invention. Usually, the bead has a porous surface, whereby pore sizes suitable for affinity chromatography, such as apheresis, are well known to the person skilled in the art.
The bead may be of any material known to the person skilled in the art, such as agarose. The bead may further be of any size the known to the person skilled in the art to be suitable for the affinity chromatography of proteins, such as microbeads. Further, the bead may bind or interact with the VEGF dimer molecule as described for the first or second aspect of the invention. Moreover, the bead may bind or interact with the matrix used in the first or third aspect of the invention, which itself binds or interacts with the VEGF dimer molecule as described for the first or second aspect of the invention. In general, each binding VEGF dimer molecule or matrix molecule that is attached to the bead may be assumed to bind in a 1:1 ratio with the solute sample sent through the column that needs to be purified or separated.
In a seventh aspect, the invention relates to a method for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood comprising incubating the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention with the blood and separating sFlt-1 from the blood and/or releasing VEGF from complexes with sFlt-1 into the blood.
In the method according to the seventh aspect of the invention, the blood may be derived from a patient before separating sFlt-1 from the blood and/or releasing VEGF from complexes with sFlt-1 into the blood. Moreover, in the method according to the seventh aspect of the invention, the blood may be returned to the patient’s blood circulation after separating sFlt-1 from the blood and/or releasing VEGF from complexes with sFlt-1 into the blood. Preferably, the method according to the seventh aspect of the invention is applied in the context of an apheresis, e.g. as described above in the context of the fourth aspect of the invention. All embodiments and features described above with respect to apheresis, e.g. as in the context of the fourth aspect of the invention, may also apply to this seventh aspect.
In the method according to the seventh aspect of the invention, sFlt-1 may be separated from blood and/or VEGF may be released from complexes with sFlt-1 into blood until the sFlt-⅟PIGF ratio in the blood decreases, preferably, until the sFlt-⅟PIGF ratio in the blood is <110, more preferably <85, especially more preferred <50 and most preferred <38.
Moreover, in the method according to the seventh aspect of the invention, sFlt-1 may be separated from blood and/or VEGF may be released from complexes with sFlt-1 into blood until the blood of the patient has amounts of sFlt-1 and/or VEGF in blood that are comparable to those of pregnant women without showing symptoms of preeclampsia.
Further, all embodiments and features described above with respect to the first, second, third and/or fourth aspect of the invention may also apply to this seventh aspect.
Moreover, the invention further relates to a method for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood comprising incubating the column obtainable by the method according to the third aspect of the invention with the blood and separating sFlt-1 from the blood and/or releasing VEGF from complexes with sFlt-1 into the blood.
In an eight aspect, the invention relates to the use of the column according to the first aspect of the invention or the VEGF dimer molecule according to the second aspect of the invention for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood.
Preferably, the use of the eight aspect of the invention is in the context of an apheresis, e.g. as described above in the context of the fourth aspect of the invention. All embodiments and features described above with respect to the first, second, third, fourth and/or seventh aspect of the invention may also apply to this eighth aspect.
Further, the invention relates to the use of the column obtainable by the method according to the third aspect of the invention for separating sFlt-1 from blood and/or releasing VEGF from complexes with sFlt-1 into blood.
The invention is not limited to the particular methodology, protocols and reagents described herein because they may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Similarly, the words “comprise”, “contain” and “encompass” are to be interpreted inclusively rather than exclusively.
Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods, and materials are described herein.
The present invention is further illustrated by the following Figures and Examples, which are intended to explain, but not to limit the invention, and from which further features, embodiments and advantages may be taken. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to the person skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is thus to be understood that such equivalent embodiments are to be included herein.
For cloning scVEGF165 and scPIGF, uncloned, double-stranded linear DNA fragments containing represented nucleotide sequence were customized and ordered in the form of GeneArt String DNA fragment (ThermoFisher scientific, Germany). The designed sequence bears two identical amino acid sequence of monomeric VEGF165 except the second monomer lacking the signal peptide sequence. Two monomers are being connected to each other using a linker with amino acid sequence of GSTSGSGKSSEGKG (showed as underlined letters below). DNA fragments were amplified using primers (see Table 1) and cloned (using restriction enzymes Nhel and BamHI) into the sleeping beauty transposon expression vector, carrying an N-terminal strep tag. Table 1: Primer list used for cloning scVEGF165 and scPIGF into expression vector.
The GeneArt String DNA fragment sequences of scVEGF165 and scPIGF are as follows (linker sequence underlined):
GeneArt String DNA fragment sequence of scVEGF165 (SEQ ID NO: 7):
GeneArt String DNA fragment sequence of scPIGF (SEQ ID NO: 8):
Protein sequence scPIGF (linker underlined) (SEQ ID NO: 9):
For the production of recombinant protein stable HEK293T Epstein-Barr virus nuclear antigen (EBNA) cell lines were generated employing the sleeping beauty transposon system and the protein was purified as previously described (Agarwal et al., 2012). Briefly, the vectors were transfected into the HEK293T EBNA cells using FuGENE® HD transfection reagent (Promega GmbH, Madison, USA) for the duration of 3 days with fetal bovine serum (FBS)- free medium. Supernatant of the cells was harvested on the following days, filtered and the proteins carrying the Strep tag were purified via Strep-Tactin®XT (IBA Lifesci-ence, Göttingen, Germany) resin at room temperature (RT). The proteins were then eluted by Biotin containing Tris-buffered saline (TBS)- buffer (IBA Lifescience, Göttingen, Germany), and the aliquots were stored at -80° C.
The structure of monomeric and dimeric VEGF was visualized by negative staining electron microscopy as described previously (Bober et al., 2010). Briefly, samples (usual concentrations 10 - 20 nM) were incubated on carbon-coated grids for 1 min, washed and then stained with 0.75% uranyl formate for 1 min. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were examined in a Philips/FEI CM 100 TWIN transmission electron microscope operated at 60 kV accelerating voltage. Images were recorded with a side-mounted Olympus Veleta camera with a resolution of 2048 x 2048 pixels (2 k x 2 K) and the ITEM acquisitions software.
HUVEC passage 3 were cultured on collagen II coated 6 well plate with Endopan 3 kit medium containing 5% FBS, hydrocortisone, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, hEGF, GA-1000 (Pan biotech, Bavaria, Germany) until they reached 80% confluency. The cells were starved with 3% FBS and no additive growth factor supplement for 24 hours. On the next day, the cells were stimulated with the recombinant proteins as indicated. After incubation for 10 minutes at 37° C., the cells were directly lysed on the plate using 30 µl 2x Laemmli buffer, and loaded on a sodium dodecyl sulfate (SDS) polyacrylamide gel, followed by western blot.
ELISA style binding assays were performed as described previously (Agarwal et al., 2012). In brief, 10 µg/ml of recombinant VEGF, PIGF and their mutant variants were individually immobilized together with bovine serum albumin (BSA) on 96 well plate (Transparente Immuno Standardmodule, Thermo scientific, Denmark), while matrix loaded only with BSA served as a control. A binding saturation curve was generated by applying increasing concentrations of VEGFR1/R2 trap ranging from 0,1 to 750 nM, followed by detection of the ligand-receptor complex using a horseradish peroxidase (HRP)-conjugated antibody against the mouse Fc domain of VEGFR1/R2 trap. Luminescence was quantified at optical density (OD) 450 nm using a by spectrophotometer (Thermo ScientificMultiskan GO, Finland).
Human sFlt-1, free VEGF, and PIGF levels were quantified using specific commercial ELISA kits (R&D Systems, Minneapolis, USA). The assays were performed as specified by the manufacturer. Fluorescence was quantified using a spectrophotometer (Thermo ScientificMul-tiskan GO, Finland). Data was analyzed with GraphPad prism 7 software (GraphPad Soft-ware Inc., La Jolla, CA, USA).
The immobilization of the recombinant protein on Strep-Tactin®XT matrix (IBA Lifescience, Göt-tingen, Germany) was performed on Polyprep® chromatography columns (Bio-Rad laboratories, USA) according to the manufacturers’ instructions.
The recombinant proteins containing a Strep tag, were incubated with the recommended amount of resin in TRIS 50 mM, NaCl 150 mM, pH=8 overnight. For immobilization of recombinant protein on Cyanogen bromide activated resin (Merck, Germany) on Polyprep® chromatography columns (Bio-Rad laboratories, USA), the proteins where dialyzed in coupling buffer containing 100 mM NaHCO3, 500 mM NaCl, pH=8.3 for 2 days. The beads were activated with 30 ml cold 1 mM HCl for 15 min. The recommended amount of resin was incubated with the dialyzed proteins overnight at 4° C. The unbound surface of the beads was blocked by Tris-HCl 0.1 M for 2 hours at RT.
For the immobilization of recombinant protein on an agarose matrix (AminoLink™ Plus Immobilization Kit, Thermo Fisher scientific, USA), the manufacturers’ instruction was followed. Briefly, the recombinant proteins were incubated in coupling buffer pH=10, added to the resin and incubated for 4 hours with 50 mM NaCNBH3 in coupling buffer pH=7.2. Blocking of the unbound surface of the resin was performed by incubation with 50 mM NaCNBH3 in quenching buffer. In all cases, the protein concentrations were measured before and after the coupling to determine the coupling efficiency. An equal concentration of around 500 mg/ml from each recombinant protein was used for immobilization to the corresponding resin.
For the assessment of the longitudinal stability of the scVEGF165 agarose column, 1.8 ml of VEGF coupled agarose resin was generated, divided into 6 aliquots of 300 µl and stored at 4° C. Adsorption studies were performed after 1, 15, 30, 60, and 90 days.
Strep-tagged proteins were precipitated from the flow through after apheresis treatment of serum samples by adding Strep-Tactin®XT (IBA Lifescience) beads. For the positive control, recombinant strep-tagged VEGF was added to the serum flow through. After washing the beads twice with TRIS 50 mM, NaCl 150 mM, pH=8, 30 µl 2 x Laemmli buffer was directly added to the beads followed by SDS polyacrylamide gel electrophoresis, and immunoblot blot using Strep-tag specific antibody.
Written informed consent was obtained from all participants according to protocols 10-238 and 09-258 as reviewed and approved by the ethics committee of the University Hospital Cologne. Preeclampsia was defined as new onset of hypertension >140/90 mmHg, proteinuria >0.3 g/g Creatinine, and sFlt-1/PIGF > 85 at EGA ≤32 weeks.
Intermolecular disulfide bonds stabilize the structure of VEGF and PIGF homodimers and unfold the receptor binding sites. These unique properties could be utilized to engineer higher order multimers of VEGF and PIGF with enhanced binding affinity to sFlt-1.
Expression constructs containing a VEGF165 lacking the N-terminal sequence followed by a short inert 14 amino acid linker and a second VEGF165 lacking the N-terminal signal peptide (single chain VEGF dimers = scVEGF165) equipped with a cleavable Strep-tag® for purification (
Monomeric wildtype VEGF165 (moVEGF165, full-length VEGF165 lacking the N-terminal sequence), and scVEGF165 constructs were cloned as detailed above (see also Table 1), expressed in human embryonic kidney cells (HEK 293T), and the protein was purified under physiologic conditions using Strep-TactinⓇ technology (
To visualize the quaternary molecular structure of scVEGF165 in comparison to moVEGF165, negative staining electron microscopy was employed (
In addition to moVEGF165 and scVEGF165, monomeric PIGF (moPIGF) and single chain PIGF dimers (scPIGF) constructs were generated and expressed and tested as previously described (
Subsequently, binding characteristics of moVEGF165, scVEGF165, moPIGF and scPIGF were explored. To screen for strong interactors, the binding affinity and static binding capacity of VEGF and PIGF constructs to VEGF-trap were quantified as the equilibrium dissociation constant (Kd) by ELISA based serial dilution experiments and protein loading in high excess, respectively. The VEGF-Trap as used in the present application is a chimeric protein containing the second binding domain of the VEGFR-1 receptor and the third domain of the VEGFR-2 receptor fused to the Fc segment of a human IgG backbone resulting in a very high VEGF binding affinity (Kd≈1 pM).
As expected from previous reports, moVEGF165 displayed higher affinity (2.6 fold) as compared to moPIGF (Christinger et al., 2004). Strikingly, scVEGF165 however showed 11% higher affinity as compared to moVEGF165. Interestingly, affinity of scPIGF was not significantly different compared to moPIGF (
Results: It is a very surprising finding that the binding affinity of scVEGF165 to VEGF-trap is 11% higher in comparison to that moVEGF165, especially in light of the fact that the binding affinity of scPIGF in comparison to moPIGF is approximately equal. Further, the surprisingly large variation in binding capacity found in the different constructs is remarkable.
To analyze coupling efficiency of recombinant proteins to different resins, the recombinant protein concentration before and after coupling to the resin was determined. The results are shown in Table 2.
Table 2: Representative data showing recombinant protein concentration before and after coupling to resin, to determine coupling efficiency of recombinant proteins to different resin.
To optimize the dynamic binding characteristics of the apheresis columns, sFlt-1 adsorption from human serum by immobilized moPIGF and moVEGF165 was tested using Streptactin XT®, Cyanogen bromide activated sepharose or aldehyde-activated agarose for immobilization of the ligand. Significant adsorption of sFlt-1 was noted for all resins on both moPIGF- as well as moVEGF165-based columns. However, the largest reduction of 86.16 % sFlt-1 in a single run was achieved with columns where scVEGF165 was immobilized on agarose matrix (
Results: Binding of a ligand to an agarose matrix led for all ligands (moPIGF, moVEGF165 and scVEGF165) to the largest adsorption of sFlt-1 from human serum in comparison to binding to a Streptactin XT® matrix or a sepharose matrix. Moreover, sFlt-1 adsorption from human serum was significantly higher for scVEGF165 in comparison to moPIGF or moVEGF165 (with moVEGF165 having a slightly better sFlt-1 adsorption from human serum in comparison to moPIGF). The largest reduction of 86.16 % sFlt-1 in a single run was achieved with columns where scVEGF165 was immobilized on agarose matrix. Moreover, the examination of longitudinal stability of scVEGF165 agarose column revealed only a slight decrease in binding sFlt-1.
In a next step, the efficacy of sFlt-1 reduction from patient serum was assessed for the different VEGF- and PIGF-variants immobilized on agarose apheresis columns.
Mini columns carrying aldehyde-activated agarose were equally loaded with VEGF- or PIGF-variants (Table 2). In addition, one antibody-based apheresis column was generated to provide a reference control. sFlt-1, VEGF-, and PIGF-concentrations were determined in a serum sample of a patient with preeclampsia using commercial ELISA kits before and after single runs over a control column or columns equipped with the respective VEGF- or PIGF-variant or Flt-1 specific antibody. Whereas no difference between sFlt-1-, PIGF-, and VEGF-concentrations was noted after running the sample over the control column, sFlt-1 levels were reduced by varying amounts depending on the specific ligand (
Strikingly, in the adsorption experiments employing patient serum, the modified single chain VEGF-dimers (scVEGF165) boasted sFlt-1 reduction to 89.87% in a single run (
The notion that increased VEGF or PIGF levels in the flow through might represent leakage of recombinant protein from the column was invalidated by purification of Strep-tagged proteins from the flow through of columns carrying Strep-tagged VEGF or PIGF after passage of serum samples. For all constructs tested, no leaked protein was detected in western blot analysis employing Strep-specific primary antibody (
Results: All columns with immobilized VEGF and PIGF variants as well as with the Flt-1 specific antibody reduced sFlt-1 in the sample. The reduction of sFlt-1 was greatest for scVEGF165 columns (p<0.0001), all other VEGF and PIGF variants as well as the Flt-1 specific antibody reduced sFlt-1 less efficiently (
The yield of sFlt-1 reduction and VEGF-/PIGF-release by scVEGF165 columns was substantiated in serum samples of 10 independent patients at different gestational ages with clinically suspected preeclampsia and high sFlt-⅟PIGF ratios.
Results: In all patient samples, sFlt-1 reduction of 88% (mean) (median 92.2%; SD 5.27) (
Apheresis systems employing Flt-1 specific antibody reduce sFlt-1 levels but do not liberate endogenous PIGF or VEGF (
In general, the unique characteristic of scVEGF165-based apheresis compared to antibody-mediated apheresis and moVEGF- or PIGF-based approaches is the enhanced affinity for sFlt-1 and thus the capability to efficiently release VEGF and PIGF (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 20164619.7 | Mar 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057019 | 3/18/2021 | WO |
