# Patent

## Title
Reference, calibrator, or control composition of PIGF-1 and sFlt-1

## Abstract

Disclosed herein are compositions and methods comprising two or more proteins in which at least one of the proteins has been altered to reduce their mutual recognition and binding. Such compositions are useful as reference, calibrators or controls in methods and assays for determining the amount of one or more of the proteins that may be present in a sample of interest or in confirming the presence of one or more of the proteins in the sample. More particularly, it relates to compositions and methods comprising altered placental growth factor-1 (PlGF-1) and soluble fms-like tyrosine kinase (sFlt-1) and methods for determining the amount or confirming the presence of sFlt-1 and/or PlGF-1 in a sample of interest.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/019,443, filed Jan. 7, 2008.


FIELD OF THE INVENTION
The present invention relates to compositions and methods comprising two or more proteins altered to prevent their mutual recognition and binding. The compositions can be used as reference, calibrator or control in analytical assays capable of detecting both altered and unaltered or native forms of one or more of the proteins.
BACKGROUND OF THE INVENTION
Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality. Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The symptoms of pre-eclampsia typically appear after the 20th week of pregnancy.
Development of a fetus and placenta is mediated by several growth factors. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, and angiogenic inducer. VEGF mediates vascular permeability and has been shown to be involved in glomerular capillary repair. VEGF binds as a homodimer to one of two homologous membrane-spanning receptor tyrosine kinases, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR).
Placental growth factor (PlGF) is a VEGF family member that is also involved in placental development. PlGF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PlGF binds as a homodimer to the Flt-1 receptor, but not the KDR receptor. Both PlGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.
A soluble form of the Flt-1 receptor (sFlt-1) has been identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placental tissue. sFlt-1 is a splice variant of the Flt-1 receptor which lacks the transmembrane and cytoplasmic domains. sFlt-1 binds to VEGF with high affinity but does not stimulate mitogenesis of endothelial cells. sFlt-1 is believed to act as a “physiologic sink” to down-regulate VEGF signaling pathways. Regulation of sFlt-1 levels therefore works to modulate VEGF and VEGF signaling pathways. Careful regulation of VEGF and PlGF signaling pathways is critical for maintaining appropriate proliferation, migration, and angiogenesis by trophoblast cells in the developing placenta.
A single gene codes for human PlGF. However, splicing of the mature PlGF mRNA results in three different length isoforms: PlGF-1 (PlGF131), PlGF-2 (PlGF152), and PlGF-3 (PlGF203). Another variant, PlGF-4, has been reported (Yang, et al, J Reprod Immunol, v 60, p 53-60, 2003). PlGF is secreted as a glycosylated homodimer.
Recently it has been shown that sFlt-1 and PlGF may be used individually or in combination as biomarkers to predict, diagnose, or monitor pre-eclampsia (Levine et al, NEJM, v 350, p 672-683, 2004).
The amino acid sequence of mature human PlGF-1, amino acid residues 1-132, has been published and is available from the Protein Data Bank identified as PDB 1FZV (Iyer, et al, J. Biol Chem, v 276, p 12153-12161, 2001). This sequence is identified herein as SEQ ID NO:1:










MLPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER




 



LVDVVSEYPS EVEHMFSPSC VSLLRCTGCC GDENLHCVPV



 



ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM



 



KPERCGDAVP RR






Diagnosis of an individual at risk for, or having pre-eclampsia may be made by determining the presence or amount of vascular endothelial growth factor, particularly PlGF, and/or receptor tyrosine kinase, particularly, sFlt-1 in a biological sample (such as urine, whole blood, serum, plasma, saliva, and so forth) taken from the individual. In analytical assays reference, calibrator and control compositions are essential for purposes of determining the amount or confirming the presence of a target analyte and, for establishing accuracy and precision of the analytical assay. The preparation of such compositions in liquid or dry form usually doesn't present difficulties if the analyte is readily available, soluble in an appropriate solvent—usually aqueous for biological analytes, stable, and does not interact deleteriously with other components that may be present in the composition. As noted above, PlGF binds sFlt-1 to form a stable association complex. As a result compositions comprising the native proteins together in independent amounts suitable for use as a reference, calibrator or control in analytical assays to detect PlGF or sFlt-1 or both PlGF and sFlt-1 cannot be prepared. Although compositions comprising the individual, separated proteins may be prepared it would be advantageous to be able to prepare compositions comprising both proteins together. Thus, a need exists for reference, calibrator or control compositions comprising these proteins together in known and independent amounts. This need has been met with the present invention.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a composition comprising two or more proteins, one or more of the proteins having been altered to sufficiently reduce or substantially prevent or eliminate mutual recognition and binding. Such a composition is useful as a reference, calibrator or control in analytical assays for one or more of the proteins in the composition.
Considering for clarity two proteins unaltered/native proteins A and B which form a non-covalent association complex, the term “substantially prevent or eliminate their mutual recognition and binding” means that in an assay to determine their mutual binding, binding of altered A to unaltered/native B, or binding of unaltered/native A to altered B, or binding of altered A to altered B is not detectable, or barely detectable, or the mutual affinity as measured quantitatively by determination of affinity constants is less than approximately 10% of that observed for unaltered/native A and unaltered/native B. The term “sufficiently reduce” means that mutual binding occurs, but it has been reduced to a degree that is acceptable for a particular application.
Consider a case where the presence or amount of unaltered or native protein A is to be determined in an analytical assay, which assay utilizes one or more receptors specific for epitopes of protein A. And consider that protein A has been altered to reduce or substantially eliminate binding to protein B. Although protein A has been altered the epitopes remain intact or acceptably intact such that they retain their ability to recognize and bind the receptors. Thereby, altered protein A is acceptable for use in calibrating the assay, confirming the presence of unaltered/native protein A in a sample, or for verifying the accuracy and precision of the assay for unaltered/native protein A. Thus, in general, receptors are capable of recognizing and binding both altered and unaltered/native forms of a protein. Analytical assays comprising receptors are usually immunoassays, which assays employ as receptors polyclonal or monoclonal antibodies, whole, polymeric and/or chimeric forms of antibodies or antibody fragments. Other kinds of receptors are also used, such as aptamers (U.S. Pat. No. 5,840,867; U.S. Pat. No. 6,207,388). In an analytical assay for the determination of unaltered or native protein B using receptors specific for epitopes of protein B, it is not necessary that epitopes of altered protein A remain intact. It is only important that mutual recognition and binding of altered protein A and protein B have been sufficiently reduced or substantially eliminated.
If both protein A and protein B have been altered to reduce or substantially eliminate their mutual recognition and binding then in an analytical assay for determination of unaltered/native protein A or an analytical assay for determining unaltered/native protein B or an analytical assay for determination of both unaltered/native proteins A and B—which assays utilize receptors specific for epitopes of protein A and receptors specific for epitopes of protein B, these epitopes in the altered proteins retain the ability to recognize and bind the receptors used in the assay. Compositions comprising both altered protein A and altered protein B together can then be used for calibrating the assays, confirming the presence of unaltered/native protein A, or unaltered/native protein B, or both unaltered/native protein A and unaltered/native protein B, and for verifying accuracy and precision of the assays.
In another aspect the present invention relates to a reference, calibrator or control composition for use in an assay for a first protein or a second protein or both first and second proteins, wherein one or more amino acids or one or more non-amino acid groups of the first protein or the second protein or the first protein and the second protein have been deleted, modified, or replaced with a different amino acid or non-amino acid group or groups thereby reducing or substantially eliminating mutual binding of the first protein and the second protein.
In another aspect the present invention relates to a composition comprising a receptor tyrosine kinase, preferably fms-like tyrosine kinase, more preferably sFlt-1 and a vascular endothelial growth factor either or both altered, by amino acid or glycosyl deletion, modification or replacement. The vascular endothelial growth factor may be a placental growth factor and preferably, PlGF-1. A preferred composition comprises sFlt-1 and altered PlGF-1 having alanine in place of:
a) proline at position 25 of SEQ ID NO:1, or
b) glutamine at position 27 of SEQ ID NO:1, or
c) cysteine at position 60 of SEQ ID NO:1, or
d) aspartate at position 72 of SEQ ID NO:1 or
e) glutamate at position 73 of SEQ ID NO:1, or
f) asparagine at position 84 of SEQ ID NO:1, or
g) proline at position 98 of SEQ ID NO:1, or
h) tyrosine at position 100 of SEQ ID NO:1, or
altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, or any combination of the alanine replacements in a) to h) and the glycine replacement.
A preferred composition comprises sFlt-1 and altered PlGF-1 having alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.
Another preferred composition comprises sFlt-1 and altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.
In another aspect the present invention relates to a method for calibrating an assay for a protein in a sample comprising the steps of:

    
    
        1) contacting a composition as described above comprising known amounts of the protein with a receptor specific for a first epitope of the protein, thereby forming a complex comprising the receptor and the protein of the composition;
        2) contacting the complex formed in step 1) with a labeled receptor specific for a second epitope of the protein, thereby forming a complex comprising receptor, the protein of the composition, and labeled receptor;
        3) detecting a signal from bound labeled receptor or a signal from free labeled receptor; and,
        4) associating the signal from free or bound labeled receptor with the known amounts of the protein in the composition.
    
    


In another aspect the invention relates to a method for calibrating an assay for a protein in a sample comprising the steps of:

    
    
        1) contacting a composition as described above comprising known amounts of the protein with an immobilized receptor specific for a first epitope of the protein, thereby forming a complex comprising immobilized receptor and the protein of the composition;
        2) contacting the complex formed in step 1) with a labeled receptor specific for a second epitope of the protein, thereby forming a complex comprising immobilized receptor, the protein of the composition and labeled receptor;
        3) separating bound labeled receptor from free labeled receptor;
        4) detecting a signal from bound labeled receptor or a signal from free labeled receptor; and,
        5) associating the signal from free or bound labeled receptor with the known amounts of the protein in the composition.
    
    


In yet another aspect the present invention relates to a method for calibrating an assay for a receptor tyrosine kinase and/or a vascular endothelial growth factor in a sample comprising the steps of:

    
    
        1) preparing a composition comprising a known or pre-determined amount of the receptor tyrosine kinase and a known or pre-determined amount of the vascular endothelial growth factor either or both altered as described above to reduce or substantially eliminate their mutual recognition and binding;
        2) contacting the composition with a receptor specific for a first epitope of the receptor tyrosine kinase and/or a receptor specific for a first epitope of the vascular endothelial growth factor, thereby forming first complexes of receptor specific for the first epitope of the receptor tyrosine kinase and receptor tyrosine kinase and/or receptor specific for the first epitope of the vascular endothelial growth factor and endothelial growth factor;
        3) contacting complexes formed in step 2) with a labeled receptor specific for a second epitope of the receptor tyrosine kinase and/or a labeled receptor specific for a second epitope of the endothelial growth factor; thereby forming second complexes comprising receptor specific for the first epitope of receptor tyrosine kinase, receptor tyrosine kinase and labeled receptor specific for the second epitope of the receptor tyrosine kinase and/or receptor specific for the first epitope of endothelial growth factor, endothelial growth factor and labeled receptor specific for the second epitope of endothelial growth factor;
        4) separating bound labeled receptor specific for receptor tyrosine kinase from free labeled receptor specific for receptor tyrosine kinase and/or bound labeled receptor specific for endothelial growth factor from free labeled receptor specific for endothelial growth factor;
        5) detecting a signal from bound labeled receptor specific for receptor tyrosine kinase or a signal from free labeled receptor specific for receptor tyrosine kinase; and/or,
        6) detecting a signal from bound labeled receptor specific for endothelial growth factor or a signal from with free labeled receptor specific for endothelial growth factor; and,
        7) associating the signal from free or bound labeled receptor specific for receptor tyrosine kinase and/or the signal from free or bound labeled receptor specific for endothelial growth factor with the known amounts of receptor tyrosine kinase and/or endothelial growth factor in the composition.
    
    


In a preferred embodiment the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have alanine in place of:
a) proline at position 25 of SEQ ID NO:1, or
b) glutamine at position 27 of SEQ ID NO:1, or
c) cysteine at position 60 of SEQ ID NO:1, or
d) aspartate at position 72 of SEQ ID NO:1 or
e) glutamate at position 73 of SEQ ID NO:1, or
f) asparagine at position 84 of SEQ ID NO:1, or
g) proline at position 98 of SEQ ID NO:1, or
h) tyrosine at position 100 of SEQ ID NO:1, or
altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, or any combination of the alanine replacements in a) to h) and the glycine replacement.
In a more preferred embodiment the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.
In another more preferred embodiment, the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.
In yet another aspect the present invention relates to a method for determining the amount or confirming the presence of a protein in a sample comprising the steps of:

    
    
        a) contacting the sample with immobilized receptor specific for a first epitope of the protein, thereby forming a first complex comprising immobilized receptor specific for the first epitope of the protein and the protein;
        b) contacting the first complex with labeled receptor specific for a second epitope of the protein, thereby forming a second complex of receptor specific for the first epitope of the protein, the protein, and labeled receptor specific for the second epitope of the protein;
        c) separating labeled receptor that is bound in the second complex from free labeled receptor;
        d) determining a signal from labeled receptor that is bound in the second complex or a signal from free labeled receptor;
        e) contacting immobilized receptor specific for the first epitope of the protein with a composition as described above comprising a known or pre-determined amount of the protein, thereby forming a third complex comprising immobilized receptor specific for the first epitope of the protein and the protein of the composition;
        f) contacting the third complex with labeled receptor specific for the second epitope of the protein, thereby forming a fourth complex of receptor specific for the first epitope of the protein, the protein of the composition, and labeled receptor specific for the second epitope of the protein;
        g) separating labeled receptor that is bound in the fourth complex from free labeled receptor;
        h) determining a signal from labeled receptor that is bound in the fourth complex or a signal from free labeled receptor; and,
        i) comparing the signals determined in d) and h) as confirmation of the presence of the protein or as a measure of the amount of the protein in the sample.
    
    


In a preferred embodiment the protein to be determined is a vascular endothelial growth factor. In a more preferred embodiment the protein to be determined is PlGF and particularly PlGF-1 and the second protein in the composition is a receptor tyrosine kinase. In a more preferred embodiment the protein to be determined is PlGF-1 and the second protein in the composition is sFlt-1. In a more preferred embodiment the protein to be determined is PlGF-1, the second protein in the composition is sFlt-1 and PlGF-1 of the composition has been altered to comprise alanine in place of:
a) proline at position 25 of SEQ ID NO:1, or
b) glutamine at position 27 of SEQ ID NO:1, or
c) cysteine at position 60 of SEQ ID NO:1, or
d) aspartate at position 72 of SEQ ID NO:1 or
e) glutamate at position 73 of SEQ ID NO:1, or
f) asparagine at position 84 of SEQ ID NO:1, or
g) proline at position 98 of SEQ ID NO:1, or
h) tyrosine at position 100 of SEQ ID NO:1, or
altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, or any combination of the alanine replacements in a) to h) and the glycine replacement.
In a more preferred embodiment the protein to be determined is PlGF-1, the second protein of the composition is sFlt-1 and PlGF-1 of the composition comprises alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.
In another more preferred embodiment, the protein to be determined is PlGF-1, the second protein of the composition is sFlt-1 and PlGF-1 of the composition comprises glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates ELISA-based binding of PlGF-1 variants to the soluble portion of Flt-1. Binding of PlGF-1 variants to human Flt-1, coated at 0.5 μg/ml on a 96-well plate, was performed using increasing concentrations of soluble proteins ranging between 1 and 16 ng/ml. Wild type PlGF-1 was used as a positive control.
FIG. 1B illustrates ELISA-based binding of PlGF-1 variants to the soluble portion of Flt-1. Percentage of binding of PlGF-1 variants at a concentration of 8 ng/ml calculated with respect to the binding of wt PlGF-1. The results shown represent the average of three independent experiments.
FIG. 2 illustrates the purity of three PlGF recombinant proteins. Silver stain (A: lanes 1, 2 and 3) and Western blot by monoclonal Rat-4 (B: lanes 4, 5 and 6). Lane assignment: M: SeaBlue ladder (Invitrogen), lane 1,4-P126(DE), lane 2,5-P126(AA) and lane 3,6-P126(GAA).



DETAILED DESCRIPTION
Although the present invention will be described in terms of certain preferred embodiments relating to pre-eclampsia and biomarker proteins sFlt-1 and PlGF-1, it should be understood that the invention relates to any protein composition and its use in which one or more component proteins of the composition have been altered to reduce their mutual recognition and binding.
Whether the pre-eclampsia biomarker proteins are determined using a single assay platform or a single kit, or determined separately in independent assays or kits, it is advantageous to have a control or calibrator comprising both biomarker proteins together in the same formulation having known or pre-determined concentrations and desired concentration ratios. There are at least two problems associated with using sFlt-1 and PlGF together in native or unaltered form to prepare reference, calibrator or control compositions: firstly, sFlt-1 and PlGF bind to each other through a specific binding domain present on each protein, as already noted, and secondly, in the serum of mid- to late-term pregnant women, sFlt-1 is typically present at a significant excess relative to PlGF whether or not they are afflicted with pre-eclampsia. Unmodified or native PlGF combined and stored together with unmodified or native sFlt-1 will not serve satisfactorily in a composition used to calibrate an assay for detection of PlGF or sFlt-1 because of the nearly quantitative binding of PlGF to sFlt-1.
Amino acid changes have been made to PlGF that reduce or substantially eliminate mutual recognition and binding of sFlt-1 and PlGF (Errico et al J. Biol. Chem. 279, 43929-43939, 2004). These amino acid changes do not have a significant impact on the overall protein structure of PlGF. Binding epitopes remain intact and permit these altered proteins to be combined and stored together with sFlt-1 in a composition for use as a reference, calibrator or control for assays designed to detect unaltered or native PlGF, unaltered or native sFlt-1, or both.
Targeting amino acid modifications, deletions or replacements to PlGF in order to reduce or substantially eliminate binding to sFlt-1 has been facilitated because the amino acid sequence of PlGF and 3-D crystal structure are available.
In general it would be advantageous to know secondary, tertiary, and quaternary structures, post-translational modifications (eg phosphorylation, glycosylation, sulfation, and ubiquitination), 3-D crystal structures of binding proteins and 3-D crystal structures of the proteins engaged in their association complex. However, this information is not required in order to practice the present invention. Although modification, deletion or replacement of groups associated with post-translational modifications can be carried out, modification, deletion or replacement of one or more amino acids of one or more of the proteins that engage in mutual recognition and binding is preferred. Site-specific chemical modification of proteins is well known in the art (Techniques in Protein Modification, Lundblad R L, CRC Press, 1995; Chemical Reagents for Protein Modification, Lundblad, R L, CRC Press, 3rd Ed, 2005). Chemical/synthetic modification of amino acids can be used to practice the present invention. A preferred approach involves genetic engineering techniques. Obtaining the amino acid sequence of a protein directly is standard practice in the art. Similarly, it is standard practice in the art to obtain the amino acid sequence of a protein indirectly from the nucleotide sequence of the gene that codes for the protein. The nucleotide sequence of a gene can be readily obtained. And, when the gene is available site-directed mutagenesis can be carried out to delete, replace, or modify one or more amino acids. This can be done in a random manner or in a predetermined manner. A protein that is altered or mutated using site-directed mutagenesis can be cloned and made readily available. Protein and genetic engineering details and protocols are readily available from numerous publications and citations therein (Molecular Cloning, Sambrook J and Russell D W, Cold Spring Harbor Laboratory Press, 2002; Recombinant Gene Expression Protocols, Tuan R S ed, Humana Press, 1997; Methods in Molecular Biology and Protein Chemistry, Spangler B D, John Wiley & Sons Ltd. 2002; Genetic Engineering Fundamentals, Kammermeyer K and Clark V L, Marcel Dekker Inc, 1989; Mayo et al, Nature v 306, p 86-88, 1983; Suggs et al, Proc Nat Acad Sci USA v 78, p 6613-6617 1981; Scott et al Nature v 302, p 538-540, 1983; Helfman et al, Proc Nat Acad Sci USA, v 80, p 31-35, 1983; Young et al, Proc Nat Acad Sci USA, v 80, p 1194-1198, 1983; U.S. Pat. No. 4,237,224; U.S. Pat. No. 4,273,875; U.S. Pat. No. 4,293,652; U.S. Pat. No. 4,870,009).
The altered protein can be tested to determine if mutual recognition and binding with its partner protein(s) have been reduced or substantially eliminated. This can be carried out using experimental protocols well known in the art. The altered protein also can be tested to determine if epitopes have been sufficiently undisturbed compared with unaltered or native protein using epitope specific receptors/antibodies. Affinity can be characterized quantitatively or qualitatively. (Errico et al, J Biol Chem, v 279, p 43929-43939, 2004; Piehler et al, J Immunol Methods, v 201(2), p 189-206, 1997; Casasnovas et al, v 270, p 13216-13224, 1995; Boone et al, J Virol, v 11, p 515-519, 1973; U.S. Pat. No. 7,081,346; U.S. Pat. No. 5,324,633; U.S. Pat. No. 4,340,668; US 2005/0175999).
Whatever the nature of the group or groups (amino acids and/or non-amino acids) altered, or the nature of the protein alteration-modification (direct chemical modification-oxidation, reduction, etc), deletion or replacement of the group(s), or whether one or each of the proteins that participate in mutual recognition and binding are altered, the two important functional features are: 1) mutual recognition and binding of an altered protein to an unaltered partner protein or binding of partner proteins when each have been altered is such that mutual recognition and binding is sufficiently reduced or substantially eliminated and 2) one or more epitopes of any altered protein retain binding properties sufficiently similar or substantially identical to the epitope(s) in the unaltered or native protein if this property is required for the particular application as discussed earlier.
Example I
Human PlGF-1 and variants, sFlt-1, anti-human PlGF-1 antibodies directed to human PlGF, binding characteristics of PlGF and variants to sFlt-1, ELISA assay for determining PlGF and other materials and experimental protocols have been described by Errico et al, J Biol Chem, v 279, p 43929-43939, 2004, and are reproduced herein in part. The Errico et al reference can be consulted for details regarding cell cultures, plasmids, selection of cell lines, and other materials and experimental protocols not explicitly provided herein.
Materials
As described by Errico et al., anti-human PlGF monoclonal antibodies and human Flt-1 (Flt-1/Fc chimera) are available from R&D Systems (Minneapolis, Minn. USA). Goat anti-mouse IgG-horseradish peroxidase (HRP) is available from Santa Cruz Biotechnology (Santa Cruz, Calif. USA; www.scbt.com).
Construction of PlGF Variants
Errico et al. obtained PlGF variants using PCR techniques carried out using the plasmid named pchPlGF-1 as template and PCR was performed using complementary primers mapping the region encoding the amino acid to be mutated to alanine and bearing the specific nucleotide modification. For the preparation of the PlGF variant having the double mutation, primers carrying both mutations were utilized. Amplified DNA was purified and used to transform competent bacteria. The plasmids were sequenced in both directions using the dideoxynucleotide method. The following PlGF-1 single residues were mutated to Ala: Asn-16, Pro-25, Gln-27, Cys-60, Asp-72, Glu-73, Asn-74, Asn-84, Pro-98, and Tyr-100. The double mutant Asp 72 to Ala and Glu 73 to Ala of PlGF-1 was also generated.
Calibrator/Control Compositions Comprising Altered PlGF-1
Calibrators/controls comprising altered PlGF-1 and sFlt-1 are prepared by combining unaltered sFlt-1 with an altered PlGF-1, in particular, the double mutant in which alanines replace aspartate at position 72 of SEQ ID NO:1 and glutamate at position 73 of SEQ ID NO:1 or the triple mutant in which there is an additional mutation of glycine replacing cysteine at position 70. These may be combined individually from dry form preparations or from working aqueous stock solutions prepared using any suitable buffer at a desired pH (such as, phosphate in saline (PBS), pH 7.5) comprising any other addenda that may be useful or required—such as anti-oxidants, preservatives, etc. For illustrative purposes, the concentration of altered PlGF-1 is in the range of 0 to about 1000 pg/mL, and sFlt-1 fixed at 100 pg/mL but other concentration ranges for both may be used. The unaltered sFlt-1 is combined with altered PlGF of the double mutant or the triple mutant in PBS (10 g NaCl, 0.25 g KCl, 1.8 g Na2HPO4, 0.3 g KH2PO4, pH 7.5) to produce the following set of reference, calibrator or control materials:














Altered PlGF1 (pg/mL)
SFlt-1 (pg/mL)









∘
100



 50
100



100
100



500
100



1000 
100










ELISA For PlGF

The quantity of PlGF in a sample of serum obtained from a pregnant woman is determined using an ELISA for PlGF. The ELISA (described in detail below) is calibrated using the set of solutions comprising altered PlGF-1 and sFlt-1 described above. The signal observed for each PlGF-1 level of the set is associated with the concentration of altered PlGF-1. The association can be represented in graphic form or correlated using appropriate statistical and mathematical calibration methods. The signal observed in the ELISA assay using the serum sample is compared with the calibration graph to determine the concentration of PlGF in the sample or transformed into concentration units using the established mathematical association.
The ELISA is carried out as follows: for determination of PlGF in a sample, one anti-human PlGF-1 monoclonal antibody at 1 μg/ml in PBS is used to coat a 96-well plate at 100 μl/well and incubated overnight at 4° C. The wells are washed once with PBS containing 0.05% TWEEN 20 (PBT) and non-specific binding sites are blocked by introducing 1% bovine serum albumin in PBS at 280 μl/well and incubation for 3 h at room temperature (RT). The wells are aspirated and kept in the cold until use. During the assay, 100 μl of each calibrator level or serum sample is appropriately diluted in PBET (PBS containing 0.1% bovine serum albumin, 5 mM EDTA, 0.05% Tween 20) and incubated for 1 hour at 37° C. The wells are washed five times by PBT and another anti-human PlGF-1 monoclonal antibody (this one HRP conjugated) diluted in PBET at 37 ng/ml, is added to the wells and incubated for 1 h at 37° C. The wells are washed five times with PBT and 100 μl of HRP substrate composed of 1 mg/ml of orthophenylenediamine in 50 mM citrate phosphate buffer, pH 5 and 0.006% H2O2 is added and incubated for 30 min in the dark at RT. The reaction is stopped by adding 25 μl/well of 4 N H2SO4, and the signal absorbance is measured at 490 nm on a microplate reader.
Comparison of Altered PlGF-1 and Unaltered PlGF Binding to sFlt-1
Errico et al. has described the experiment to determine the binding of altered PlGF-1 and unaltered/native PlGF-1 to Flt-1. Basically, a 96-well plate is coated with a soluble human Flt-1 (Flt-1/Fc chimera) at 0.5 μg/ml in PBS, pH 7.5, 100 μl/well, overnight at RT. The plate is washed five times with PBT, and after the blocking non-specific sites of wells with bovine serum albumin solution as described above, the binding reaction is allowed to proceed by adding altered PlGF-1 or unaltered/native PlGF to a well and incubating for 1 h at 37° C. and 1 h at RT. The wells are washed with PBT as described above and incubated with a biotinylated anti-human PlGF-1 polyclonal antibody, 300 ng/ml in PBET, for 1 h at 37° C. and 1 h at RT. Detection is performed as described above in the ELISA assay and the signals obtained with altered PlGF-1 and unaltered/native PlGF-1 are compared. The results obtained by Errico et al. are reproduced in FIG. 1A and FIG. 1B.
Example II
Comparing Recombinant PlGF (DE) and PlGF (AA)
Experiment Purpose:
Two recombinant PlGF proteins were evaluated (1) for their binding reactivity to monoclonal antibody specific to human PlGF, and (2) for their binding reactivity to sFlt, the formation of ligand:receptor complex.
Materials and Reagents:
(1) Recombinant PlGF:
Two versions of purified recombinant PlGF were used. The protein consists of a 21-amino-acid leader sequence that does not belong to PlGF. The leader sequence contains a “6×His” tag and a 4-amino-acid Xa recognition and cleavage site.










Leader sequence: SEQ ID NO:2:




MRGSHHHHHHGSGSGSGIEGR






The PlGF portion sequence in PlGF (DE): Amino acid sequence corresponds to wild-type PlGF amino acids 4-132 of SEQ ID NO:1, resulting in the following DE amino acid sequence:











SEQ ID NO:3:




AVPPQQWALS AGNGSSEVEV VPFQEVWGRS YCRALERLVD



 



VVSEYPSEVE HMFSPSCVSL LRCTGCCGDE NLHCVPVETA



 



NVTMQLLKIR SGDRPSYVEL TFSQHVRCEC RPLREKMKPE



 



RCGDAVPRR






The PlGF portion sequence in PlGF (AA): Amino acid sequence corresponds to wild-type PlGF amino acids 4-132 of SEQ ID NO:1 with two mutations made at amino acid positions 72 and 73 in SEQ ID NO:1, resulting in the following AA amino acid sequence:











SEQ ID NO:4:




AVPPQQWALS AGNGSSEVEV VPFQEVWGRS YCRALERLVD



 



VVSEYPSEVE HMFSPSCVSL LRCTGCCGAA NLHCVPVETA



 



NVTMQLLKIR SGDRPSYVEL TFSQHVRCEC RPLREKMKPE



 



RCGDAVPRR






(2) Recombinant sFlt:

Full length sFlt was obtained from Scios Inc. (Mountain View, Calif. USA; www.sciosinc.com) (Lot#9225-89), consists of 687 amino acids of soluble fms-like tyrosine kinase 1 (sFlt-1).
sFlt-1 sequence:








SEQ ID NO:5:



MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLH


 


LQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQAN


 


HTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTE


 


GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK


 


EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVL


 


NCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDK


 


MQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGK


 


RSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA


 


GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQ


 


ILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGN


 


RIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISF


 


YITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTM


 


HYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQK


 


KEITIRGEHCNKKAVFSRISKFKSTRNDCTTQSNVKH






(3) Monoclonal Antibody to Human sFlt-1 and to Human PlGF:















monoclonal Ab ID
Source
Cat #
Lot
Clone



















RD-1 mouse anti-sFlt
RD Sys

CGG07605B
49560


RD-2 mouse anti-sFlt
RD Sys

BYC01605A
49543


Rat-1 rat anti-PlGF
RD Sys
n/a
1103925
358903


Rat-2 rat anti-PlGF
RD Sys
n/a
1103933
358939


Rat-3 rat anti-PlGF
RD Sys
n/a
1103931
358932


Rat-4 rat anti-PlGF
RD Sys
n/a
1103926
358905


Rat-5 rat anti-PlGF
RD Sys
n/a
1103927
358907


MS-1 mouse anti-PlGF
RD Sys
MAB264
n/a
37203









Experiment Examples:

(1) ELISA Assay-1:

    
    
        High-binding microtiter plate was coated with recombinant PlGF(DE) or PlGF(AA) at 0.5 ug/mL and blocked with BSA/PBS
        Standard ELISA procedure consists of monoclonal antibody dilution in casein/PBS; dilution of HRP conjugated donkey anti-mouse IgG or donkey anti-rat IgG at 1:3K in casein/PBS; 100 uL/well sample or conjugate volume; each step incubation at 37 C/30 min/shake; 6 times plate washing, 100 uL OPD substrate development for 25 C/30 min; 25 uL stop solution; record OD at 492 nm.
        ELISA results assay 1 are shown in Table 1.
    
    









TABLE 1







Recognition of monoclonal anti-PlGF to recombinant PlGF


(unaltered and altered)








Coated
Antibody binding activity to coated PlGF (OD)


recombinant
Monoclonal anti-PlGF ID, clone # and concentration (ng/mL)














PlGF
Ms-1
Rat1
Rat2
Rat4

Rat3
Rat5


(0.5 μg/mL)
37203
358903
358939
358905

358932
358907


















PlGF-1 (DE)
Ab
1.823
2.098
2.237
2.114
Ab
1.233
1.256


PlGF-1 (AA)
(10 ng/mL)
2.650
1.789
2.233
1.935
(100 ng/mL)
1.305
1.297









    
    
        Conclusion: All monoclonal antibodies tested reacted to both PlGF(DE) and PlGF(AA), indicating that D72/E73A mutation did not affect monoclonal antibody binding and these antibody epitope locations were not at these two mutation sites.

(2) ELISA Assay-2:

        High-binding microtiter plate was coated with recombinant PlGF(DE) or PlGF(AA) at 0.5 ug/mL and blocked with BSA/PBS
        Standard ELISA procedure consists of 1st plate incubation with diluted sFlt in casein/PBS at various concentrations; 2nd plate incubation with mixed anti-sFlt solution comprising two monoclonal antibodies of RD-1 and RD-2 each at 0.1 μg/mL; 3rd plate incubation with HRP conjugated donkey anti-mouse at IgG at 1:4K dilution in casein/PBS; and 4th plate incubation with 100 μL OPD substrate development for 30 min at 25° C. 1st, 2nd and 3rd plate incubation steps are for 15-20 min/shake at 37° C.; 6 times plate washing between each step. 25 μL stop solution after 4th incubation and record OD at 492 nm.
        ELISA results assay-2 are shown in Table 2.
    
    









TABLE 2







Binding of sFlt to recombinant PlGF (unaltered and altered)












Complex formation




Coated
of sFlt to coated PlGF



recombinant
sFlt concentration



PlGF
(ng/mL) at incubation













(0.5 μg/mL)
1800
600
200
66.67







BSA (control)
0.004
0.006
0.010
0.020



PlGF-1 (DE)
1.728
1.204
0.524
0.316



PlGF-1 (AA)
0.150
0.080
0.060
0.040







* Bound sFlt:PlGF complex were detected by mouse anti-sFlt and HRP anti-mouse conjugate






    
    
        Conclusion: sFlt formed receptor:ligand complex with coated PlGF(DE). However, such complex formation was greatly reduced with PlGF(AA) mutant, indicating that amino acid positions 72 and 73 in SEQ ID NO:1 were critical for sFlt-1 binding and complex formation.

(3) Biacore Assay:

        sFlt were immobilized on Biacore chip FC-2 via NHS/EDC coupling to a RU=6738. FC-1 was blank as negative control.
        PlGF(DE) or PlGF(AA) were injected to FC-1 and FC-2 to evaluate complex formation
        Biacore results are shown in Table 3.
    
    









TABLE 3







Biacore measurement


of sFlt:PlGF complex










recomb hu




P1GF 20 μg/mL












CM5 Chip:

PlGF (DE)
PlGF (AA)
















Bicore
FC1: blank
11
12



(RU)
FC2: sFlt
156
30




FC2 − FC1
145
18










    
    
        Conclusion: Injected PlGF(DE) bound to immobilized sFlt to form receptor:ligand complex while injected PlGF(AA) bound to immobilized sFlt poorly.
    
    


Example III
Comparison Between Wild-Type Recombinant PlGF and Two Additional PlGF Mutants
Experiment Purpose:
Three additional recombinant PlGF proteins were constructed and evaluated (1) for their binding reactivity to monoclonal antibody specific to human PlGF, and (2) for their binding reactivity to sFlt, the formation of ligand:receptor complex.
Materials and Reagents:
(1) Three additional recombinant PlGF proteins were constructed as follows:








P126(DE): recombinant PlGF wild type: SEQ ID NO:6:



MRGSAVPBQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP


 


SEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPS


 


YVELTFSQHVRCECRPLREKMKPERCGDAVGPGQIVGGVYLL






The first four amino acid residues are unrelated amino acids (MRGS); the last ten amino acids are the 10 G epitope (C terminal tag); the two amino acids preceding the 10 G epitope are also unrelated amino acids (Gly-Pro); the 126 amino acid sequence between the unrelated amino acids (i.e. beginning after MRGS and preceding GP) is the PlGF sequence identical to amino acid positions 4 to 129 in SEQ ID NO:1.









P126(AA): PlGF mutant #1: SEQ ID NO:7:



MRGSAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP


 


SEVEHMFSPSCVSLLRCTGCCGAANLHCVPVETANVTMQLLKIRSGDRPS


 


YVELTFSQHVRCECRPLREKMKPERCGDAVGPGQIVGGVYLL






same as P126(DE) except the underlined amino acids (AA) are the two mutated amino acids









P126(GAA): PlGF mutant #2: SEQ ID NO:8:



MRGSAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP


 


SEVEHMFSPSCVSLLRCTGCGGAANLHCVPVETANVTMQLLKIRSGDRPS


 


YVELTFSQHVRCECRPLREKMKPERCGDAVGPGQIVGGVYLL






same as PlGF mutant#1 P126(AA) except an additional mutation at the amino acid two before AA is mutated from C to G

All three recombinant proteins were expressed in bacteria and all form insoluble inclusion bodies. After sonication, washing with 4 M urea in PBS and 2M urea in PBS, inclusion bodies were finally solubilized by 8M urea/15 mM reduced Glutathione (GSH)/50 mM Tris-HCL (pH7.8). PlGF proteins were refolded through three step dialysis: (1) 24 hours against dialysis buffer 3M urea/50 mM TRIS (pH7.5)/2 mM EDTA/0.2 M Arginine/2 mM GSH, (2) 24 hours against dialysis buffer 2M urea/50 mM TRIS (pH7.5)/2 mM EDTA/0.2 M Arginine/1.2 mM GSH/0.4 mM oxidized Glutathione (GSSG) and (3) 24 hours against dialysis buffer 0.8M urea/20 mM TRIS (pH7.5)/2 mM EDTA/0.2 M Arginine/0.48 mM GSH/0.16 mM GSSG. Refolded PlGF were further purified by loading dialyzed protein solution to an affinity column, prepared by cross linking monoclonal antibody specific to 10 G tag and CNBr-activated Sepharose 4 Fast Flow resin (GE catalogue #17-0981-01). The bound PlGF was then eluted by 40% acetonitrile. Purified PlGF proteins were finally obtained after buffer exchange to PBS.
(2) Monoclonal anti-human PlGF, monoclonal anti-human sFlt are available from R&D Systems (Minneapolis, Minn. USA); HRP conjugated donkey anti-rat IgG (Cat#712-035-150) are available from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa., USA); ELISA plates were Costar hind binding by Corning Life Sciences (Cat#2592); Electrophoresis gels NuPAGE 4-12%, PVDF transfer membrane and SeeBlue ladder were from Invitrogen (Carlsbad Calif., USA); Blocker casein/PBS and SuperSignal West Dura western blot substrate were purchased from Pierce (Rockford, Ill., USA). CNBr-activated Sepharose 4 Fast Flow resin (Cat #17-0981-01) and Silver stain kit (Cat #17-1150-01) were from GE Healthcare (Piscataway, N.J., USA)

Experiment Examples:

(1) Recombinant PlGF: FIG. 2 Shows the Purity of the Three PlGF Recombinant Proteins by Silver Stain and Western Blot.

(2) ELISA Assay-1

    
    
        High-binding microtiter plate was coated with recombinant P126(DE) or P126(AA) or P126(GAA) at 0.5 ug/mL and blocked with BSA/PBS
        Standard ELISA procedure consists of monoclonal antibody dilution in casein/PBS; dilution of HRP conjugated donkey anti-mouse IgG or donkey anti-rat IgG at 1:3K in casein/PBS; 100 uL/well sample or conjugate volume; each step incubation at 37 C/30 min/shake; 6 times plate washing, 100 uL OPD substrate development for 25 C/30 min; 25 uL stop solution; record OD at 492 nm.
        ELISA results are shown in Table 4.
    
    









TABLE 4







Recognition of monoclnal anti-PlGF to recombinant PlGF (unaltered and altered)









Antibody binding activity to coated PlGF (OD)


Coated
Monoclonal anti-PlGF ID, clone # and concentration (ng/mL)














recombinant PlGF
Ms-1
Rat1
Rat2
Rat4

Rat3
Rat5


(0.5 ug/mL)
37203
358903
358939
358905

358932
358907


















P126 (DE)
Ab at
1.023
1.368
1.497
1.634
Ab at
0.779
0.834


P126 (AA)
1 ng/mL
0.967
1.301
1.633
1.681
10 ng/mL
0.681
0.697


P126 (GAA)

1.134
1.226
1.530
1.591

0.806
0.799









    
    
        Conclusion: All monoclonal antibodies tested reacted to P126(DE), P126(AA) and P126(GAA), indicating that D72A/E73A double mutation and C70G/D72A/E73A triple mutation did not affect monoclonal antibody binding and these antibody epitope locations were not at these mutation sites.

(3) ELISA Assay-2:

        High-binding microtiter plate was coated with recombinant P126(DE), P126(AA) and P126(GAA) at 0.5 ug/mL and blocked with BSA/PBS
        Standard ELISA procedure consists of 1st plate incubation with diluted sFlt in casein/PBS at various concentration, 2nd plate incubation with mixed anti-sFlt solution comprising two monoclonal antibodies of RD-1 and RD-2 each at 0.1 ug/mL, 3rd plate incubation with HRP conjugated donkey anti-mouse IgG at 1:4K dilution in casein/PBS and 4th plate incubation with 100 uL OPD substrate development for 25 C/30 min. 1st, 2nd and 3rd plate incubation step is at 37 C/15-20 min/shake; 6 times plate washing between each step. 25 uL stop solution after 4th incubation and record OD at 492 nm.
        ELISA results are shown in Table 5.
    
    









TABLE 5







Binding of sFlt to recombinant PlGF (unaltered and altered)












Complex formation of





sFlt to coated PlGF




sFlt concentration



Coated recombinant
(ng/mL) at incubation













PlGF at (5 ug/mL)
1800
600
200
66.67

















BSA (control)
0.009
0.008
0.005
0.003



P126 (DE)
>3
1.833
0.833
0.347



P126 (AA)
0.210
0.182
0.115
0.143



P126 (GAA)
0.150
0.101
0.095
0.088







* Bound sFlt:PlGF complex were detected by mouse anti-sFlt and HRP anti-mouse conjugate






    
    
        Conclusion: sFlt formed receptor:ligand complex with coated P126(DE). However, such complex formation was greatly reduced with P126(AA) mutant and P126(GAA) mutant, indicating that amino acid position 70, 72 and 73 in SEQ ID NO:1 were critical for sFlt-1 binding and complex formation.

(4) ELISA Assay-3:

        High-binding microtiter plate was coated with recombinant sFlt at 0.5 ug/mL and blocked with BSA/PBS
        Standard ELISA procedure consists of 1st plate incubation with diluted P126(DE) or P126(AA) or P126(GAA) in casein/PBS at various concentration, 2nd plate incubation with monoclonal anti-PlGF Rat-4 solution at 0.1 ug/mL, 3rd plate incubation with HRP conjugated donkey anti-Rat IgG at 1:4K dilution in casein/PBS and 4th plate incubation with 100 uL OPD substrate development for 25 C/30 min. 1st, 2nd and 3rd plate incubation step is at 37 C/15-20 min/shake; 6 times plate washing between each step. 25 uL stop solution after 4th incubation and record OD at 492 nm.
        ELISA results are shown in Table 6.
    
    









TABLE 6







Binding of recombinant PlGF (unaltered and









Complex formation of PlGF (unaltered or altered) to


Coated
PlGF (ng/mL) at












sFlt at (0.5
1000
500
250
100
0















P126 (DE
>3
1.388
0.557
0.259
0.021


P126 (A
0.299
0.118
0.101
0.077
0.009


P126 (GA
0.119
0.117
0.069
0.088
0.051





* Bound sFlt:PlGF complex were detected by mouse anti-PlGF (Rat-4) and conjugat






    
    
        Conclusion: unaltered PlGF, P126(DE), formed ligand:receptor complex with coated sFlt. However, altered PlGF (P126(AA) and P126(GAA) failed to form such complex, indicating that amino acid position 70, 72 and 73 in SEQ ID No:1 were critical for sFlt binding and complex formation.
    
    


The description of the specific embodiments of the invention is presented for the purposes of illustration. It is not intended to be exhaustive or to limit the scope of the invention to the specific forms described herein. It will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and scope of the invention as set forth in the claims.
All patents, patent applications, and publications cited herein are hereby incorporated by reference.

## Claims

1. A reference, calibrator or control composition comprising PlGF-1 and sFlt-1, where the PlGF-1 comprises alanine in place of:
a) proline at position 25 of SEQ ID NO:1, or
b) glutamine at position 27 of SEQ ID NO:1, or
c) cysteine at position 60 of SEQ ID NO:1, or
d) aspartate at position 72 of SEQ ID NO:1 or
e) glutamate at position 73 of SEQ ID NO:1, or
f) asparagine at position 84 of SEQ ID NO:1, or
g) proline at position 98 of SEQ ID NO:1, or
h) tyrosine at position 100 of SEQ ID NO:1, or

altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, or any combination of the alanine replacements in a) to h) and the glycine replacement.

2. The composition of claim 1 wherein PlGF-1 comprises alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.

3. The composition of claim 1 wherein PlGF-1 comprises glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.