PEPTIDE AND ASSAY Technical Field The current invention relates to a method of assaying for the soluble tyrosine kinase receptor Flt-1 (sFlt-1). In particular, the invention relates to a method of assaying for sFlt-1 in mammalian plasma or serum samples.

Background to the Invention Vascular endothelial growth factor (VEGF) is a potent angiogenic regulator and is known to have anti-apoptotic as well as mitogenic activities. It is essential for the proper development of embryonic vasculature, the growth of both maternal and fetal blood vessels in the uterus, the cyclical growth of blood vessels in the female reproductive tract and the formation of capillaries during wound repair.

VEGF is also involved in abnormal angiogenesis as seen in retinopathies, rheumatoid arthritis, psoriasis and malignant cancers.

VEGF exerts these activities through two tyrosine kinase receptors namely the tyrosine kinase receptor Flt-1 and the kinase insert-domain containing receptor (KDR or Flk-1), which are predominantly localised on endothelial cells. Binding of VEGF causes receptor dimerisation and autophosphorylation for signalling.

Alternative splicing of the Flt-1 gene produces two products, one encoding the full-length receptor and the other a soluble form (sFlt-1). SFlt-1 is a selective VEGF inhibitor that binds to VEGF with high affinity, thus preventing VEGF-induced receptor phosphorylation. Because VEGF plays such an important role in normal and pathological angiogenesis, (e. g. placental VEGF overproduction in response to local hypoxia is involved in preeclampsia, which complicates 5 to 10% of all pregnancies), monitoring levels of VEGF and sFlt-1 reliably in the serum or plasma of patients suffering from cancer or other diseases, but especially in the plasma of women during pregnancy, will be of major benefit.

SFlt-1 is a heparin-binding lOOKDa protein, which selectively binds VEGF and is involved in the regulation of VEGF's biological activity. Therefore, it is important to measure and investigate levels of sFlt-1 in the bloodstream in patients suffering from diseases that involve abnormal angiogenesis, e. g.

pre-eclampsia, cancer, rheumatoid arthritis retinopathies and ovarian hyperstimulation syndrome.

Currently the only assay for sFlt-1 is an Enzyme Linked Immunosorbent Assay (ELISA). However, this is insufficiently insensitive to measure accurately the total circulating sFlt-1 in many physiological and pathological states, due to the various sFlt-1 binding complexes that can exist in the circulation.

It is an object of the invention to overcome at least some of the above problems.

Statements of Invention With this in mind, a new assay has been developed that, in one aspect, relies on the recognition of a unique peptide sequence, NI-11 (Sequence Id No 1), found within the sFlt-1 protein, by specifically produced antisera.

According to the invention, there is provided an isolated peptide comprising an amino acid sequence having at least partial homology with the sequence of Sequence ID No 1. In this specification, the term "partial homology"should be understood as meaning at least a 50% homology, suitably at least a 60% homology, typically at least a 70% homology, preferably at least a 80% homology, and ideally at least a 90% homology. Typically, the isolated peptide, and especially a carboxy terminal portion of the isolated peptide, is homologous with at least

a carboxy terminal portion of the sequence of Sequence ID No. 1, wherein the carboxy terminal portion consists of at least 4, and preferably 5 or 6, amino acids. In a preferred embodiment of the invention, the isolated peptide comprises an amino acid sequence which is homologous with that shown in Sequence ID No. 1. Thus, the invention also relates to an isolated peptide comprising, or consisting essentially of, a carboxy terminal portion consisting of a sequence selected from the group comprising: T-N-T-I ; Q-T-N-T-I ; AND R-Q-T-N-T-I.

The invention also relates to fragments or analogues of the isolated peptides of the invention.

Analogues may include moieties which mimic the structure of amino acids, or may include non-peptide bonds.

The invention provides a multiple antigenic peptide (MAP) which includes a plurality of isolated peptides of the invention. Ideally each isolated peptide consists of the amino acid sequence of Sequence ID No. 1. Preferably, each isolated peptide is attached to the MAP backbone at its amino terminal end. Suitably, the MAP backbone comprises a lysine tree, the details of which will be well known to those skilled in the art.

The invention also relates to an antibody, or anti- sera which contains antibodies, which binds specifically to a peptide of the invention.

The invention also relates to an antibody or anti- sera raised by immunising a suitable subject with either or both of the isolated peptide, or a MAP, of the invention. Preferably the antibody or anti-sera is raised by immunising a suitable subject, such as a rabbit, with such a MAP. The primary antibody of the invention may be monoclonal or polyclonal, or an anti-sera preparation. Methods for producing both monoclonal and polyclonal antibodies, and anti-sera, are well known to those skilled in the art of antibody production.

The invention also relates to a method of assaying for sFlt-1 protein which is a competitive immunoassay in which a labelled competitive ligand is employed, wherein the labelled competitive ligand comprises a peptide according to the invention.

Generally, the method comprises use of a primary antibody or anti-sera which is an antibody or anti- sera according to the invention.

In a preferred aspect, the invention provides a method of assaying for sFlt-1 protein comprising the steps of: - providing a sample to be assayed; - treating the sample to cleave any sFlt-1 protein present in the sample to produce sFlt-1 peptide fragments; - incubating the treated sample with a (primary) antibody or anti-sera of the invention and a labelled competitive ligand;

separating the (primary) antibody bound from the sample using a secondary antibody or anti-sera which specifically binds the antibody of the invention; assaying the bound antibody for labelled competitive ligand; and determining the amount of sFlt-1 protein in the sample, wherein the labelled competitive ligand comprises an isolated peptide of the invention.

Typically, the secondary anti-sera is a precipitating secondary anti-sera.

If sFlt-1 is present in the sample, the enzyme treatment will result in NI-11 peptide being released into solution. This free peptide will then compete with the labelled competitive ligand for binding sites on the primary antibody. Thus, when there is a high concentration of sFlt-1 in the sample, less labelled competitive ligand will be able to bind to the primary antibody, and thus label will be detected when the bound antibody is assayed. Likewise, when there is a low concentration of sFlt-1 in the sample, then more labelled competitive ligand will bind to the antibody, and less label will be detected when the bound antibody is assayed for label. Thus, the label of sFlt-1 present in the sample is effectively inversely proportional to the level of labelled competitive ligand which is bound to the primary antibody.

Typically, the sample to be assayed is treated with an enzyme to cleave the sFlt-1 protein. One suitable enzyme is elastase. Such enzyme treatment cleaves the sFlt-1 protein after the C-terminal amino acid residue in the NI-11 sequence and at the C-terminal end of the third amino acid residue within the sequence therefore liberating an 8-amino acid sequence homologous to the C-terminal residues of the sequence given in Sequence ID No. 1 The competitive ligand may be labelled with any suitable label such as a radioactive label or a luminescent or fluorescent label. Preferably, the ligand is labelled with radioactive label such as iodine. Suitably, the ligand is incorporated with 0. 6mCil25I.

Typically, the sample is plasma or serum, in one preferred embodiment human plasma or serum.

Generally, serum (or plasma) samples are processed to generate assayable sFlt-1 peptide fragments. The NI11 sequence is otherwise undetectable when sFlt-1 is complexed with VEGF or dimerised. After the extraction procedure samples (or standards) are incubated with the specific antisera at 4 degrees C prior to the addition of purified Iodo-radiolabelled peptide. Following a further 24 hour incubation (4 degrees C) a precipitating secondary anti-sera (SAR) is used to separate antibody-bound from free peptide (NI-11) and subsequent radiation analysis allows the

amount of peptide to be calculated and hence the total amount of sFlt-1 in the sample. This is based on standard radioimmunoassay principles where the primary anti-NI11 antibody has equal affinity for the peptide sequence whether the sequence is derived from plasma or serum sample or purified radiolabelled or unlabelled (standard) peptide is used. A primary antibody dilution is used which binds 45-50% of the radiolabelled NI-11 sequence and thus allows for a competitive equilibrium between radiolabelled and unlabelled peptide to be established. The quantity of peptide in any specimen is,. therefore, inversely propertional to the amount of radiolabelled NI-11 peptide bound to the antibody. The concentration of sFlt-1 can then be quantitatively assessed by comparing the percentage binding obtained for each sample with that of a known sFlt-1 standard concentration on a standard curve.

The invention also relates to a kit for assaying a sample for sFlt-1 protein comprising: - an antibody or anti-sera according to the invention; - a labelled competitive ligand; and - a secondary antibody specific to the antibody or anti-sera of the invention; wherein the labelled competitive ligand is an isolated peptide of the invention.

Suitably, the kit also includes means for cleaving sFlt-1 protein in the sample such as, for example, a suitable enzyme such as elastase.

In one preferred embodiment of the invention, the competitive ligand comprises a peptide having an amino acid sequence, which is at least 80% homologous with that shown in Sequence ID No. 1.

In one embodiment of the invention, there is provided a competitive immunoassay for sFlt-1 protein which employs a labelled competitive ligand, wherein the labelled competitive ligand comprises a peptide having an amino acid sequence which is at least partially homologous with that shown in Sequence ID No. 1. In one preferred embodiment of the invention, the competitive ligand comprises a peptide having an amino acid sequence which is homologous with a carboxy terminal portion of the amino acid sequence shown in Sequence ID No. 1.

The invention also relates to a method of predicting, screening for, and/or diagnosing pre- eclampsia in an individual which includes the step of assaying a biological sample from the individual for sFlt-1. Suitably, the biological sample will be assayed according to a method of the invention.

The invention also relates to the use of a kit according to the invention in (a) the diagnosis of pre-eclampsia or (b) monitoring tumour angiogenesis.

Typically, the kit of the invention comprises a container (A) which holds a predetermined concentration of an enzyme, typically elastase. A second container (B) holds a predetermined concentration of the antibody in a biological buffer. A third container (C) holds radiolabelled ligand in solution at a predetermined concentration.

A fourth contained (D) contains the secondary antibody, at a predetermined concentration. A fifth container (E) contains normal rabbit Serum and a sixth container (F) contains 4% Polyethylene Glycol.

A seventh container (G) will contain a known amount of peptide standard for dilution and construction of the assay calibration curve. Further containers may be produced to contain a negative control and several quality control peptide standards.

Biological samples are treated as follows: An aliquot from container A is added to a predetermined volume of sample to be tested and the sample is incubated at 37 degrees C for 5 Minutes. This is followed by protein extraction using alcohol (typically ethanol), whereby a predetermined volume of alcohol is added to each sample prior to centrifugation at 3000rpm for 30 minutes. The supernatant (containing the liberated NI-11 assayable sequence) is then collected and air-dried.

Prior to assay, the sample is reconstituted by the addition of a predetermined amount of physiological buffer. A known volume of extracted sample (typically 100 microlitres) is then used in the subsequent assay protocol.

An aliquot from container B is added and followed by an overnight incubation at 4 degrees C. An aliquot from container C is then added prior to a further incubation (typically 24 hours) at 4 degrees C.

Separation of antibody bound and unbound is achieved by the addition of aliquots from containers D, E and F to the sample. This is followed by the mixing of each sample and a 90 minute incubation at 4 degrees C. Thereafter, samples are centrifuged at 3000rpm for 30 minutes for 30 minutes and the supernatant decanted. The radiation in each precipitate is then analysed using a gamma counter. The degree of radiolabelled peptide binding to the antibody is calculated and compared to the standard curve, which is established in parallel with the specimen analysis.

Detailed Description of the Invention The invention will be more clearly understood from the following description of an embodiment thereof, with reference to the accompanying figures, in which: Figure 1 is an antibody titre curve showing the amount of sFlt-1 labelled peptide bound to the sFlt- 1 antibody after immune precipitation with a secondary antibody; and Figure 2 is a diagrammatic representation of the experiments involved in the production of the sFlt-1

peptide and its Radiolabelling with Iodine125 and the subsequent peptide antibody production.

As shown in Figure 2, the 2 Dimensional structure and full amino acid sequence of human Flt-1 is known, from which an 11 amino acid peptide sequence (NI-11) has been identified within the VEGF binding site. The 11 amino acid sequence is identified below: Sequence ID No. 1 N-Y-L-T-H-R-Q-T-N-T-I 1 10 Through extensive screening of possibly useful peptides, the above peptide, and peptides including this sequence, were found to be the most suitable due to their relatively exposed surface position on the sFlt-1 VEGF binding site.

The peptide was synthesised using standard FMOC chemistry in 25umol scale set-ups, using the Synergy 432A peptide synthesizer.

Standard biochemical analysis of the thus formed peptide was carried out by reverse phase HPLC using a C18 Hi-pore reversed phase column and the peptide's identity was verified by electrospray mass spectroscopy (ES-MAS) using a Finnigan LCQ Ion Trap Mass Spectrometer. A multiple antigenic peptide (MAP) was also synthesised and rabbits were

thereafter immunised with this sFlt-1 MAP peptide.

Linear and MAP peptides were freeze-dried and stored at-20°C until required.

Production of sFlt-1 peptide (NI-11) antibodies The MAP NI-11 sequence was equilibrated to room temperature (-25°C). The NI-11 MAP complex (4mg) was dissolved in 2ml of distilled H20. Complete Freund's adjuvant (2ml) was added and the solution emulsified, prior to primary immunisation. A further 6 mg of MAP complex was dissolved in 3mls of distilled H20, divided into 0. 5mls lots and stored at-20°C for boosts.

Rabbits were injected subcutaneously in 3 to 4 different sites. Each received a primary immunisation (Img) of peptide/MAP emulsified in complete Freund's adjuvant. After 6 weeks rabbits were injected with 50Ag peptide/MAP emulsified in incomplete: complete Freund's adjuvant (9: 1 [v/v]), and continued to be boosted at 4-week intervals.

After each boost injection, blood (approx. 20ml) was taken from the lateral ear vein. The blood was stored overnight at 4°C before the serum was separated by centrifugation at 1500rpm for 30 min at 4°C. The serum from each bleed was stored in aliquots at-20°C.

The presence of sFlt-1 (NI-11) antibodies in the collected sera was confirmed by incubating various

serum dilutions with the corresponding radiolabelled (NI-11) peptide. sFlt-1 peptide radio-labelling 0. 8mCi Na125I was incorporated into the purified sFlt-1 peptide (NI11) was incorporated with using the Iodogen method. Peptide (20 microgram) was dissolved in iodination buffer (0.2M phosphate pH 7.4) prior to addition of Na125I to the reaction vial. The reactants were mixed carefully at regular intervals during a 15 minute reaction time. The reaction was terminated by the addition of 0. 05% TFA in water. Radiolabelled and unlabelled NI-11 peptides were separated by reverse phase HPLC and the purified sFlt-1 (NI-11) peptide radiolabel was collected and stored in acid alcohol at-20 degrees C.

A solution of sFlt-1 labelled peptide was incubated with sFlt-1 antibodies @ 4°C for 48 hrs and the Radioimmunoassay (RIA) antibody titre was obtained (Fig. 1).

RIA Standard Curves A protein free serum medium was used as buffer in all assays. Peptide standards ranging from 0- 500pg/ml [0-368pmol/L] incubated with NI-11 peptide antibody overnight @ 4°C. Radiolabelled NI-

11 peptide was then added followed by a further 24 hour incubation. Separation of bound and free was achieved by immunoprecipitation of the primary antibody complex using a secondary antibody. The secondary antibody was typically sheep anti-rabbit and was used at a 1/25 dilution. Normal rabbit serum [1/3000] and 4% PEG were also typically used in the separation procedure.

RESULTS SFlt-1 antibodies were produced and used at a working dilution of approximately 1/500,000 (see Figure 1).

Standard curve 10% fall = 1. 8pmol/l; ID50 = 7pmol/l sFlt-1 Assay Sensitivity: The sensitivity of the assay can be increased by using a greater sample volume (200 microlitres).

However, the routine assay using a 100-microlitre sample is typically optimum and has a maximum sensitivity of 0. 7pmol/L sFlt-1. When 200 microlitres of sample is used, a maximum sensitivity of 0. 3pmol/L is possible.

Coefficients of Variation: 1. Peptide Standards

Various NI-11 peptide concentrations were analysed in the assay and the intra-assay coefficient of variation for each was calculated: NI-11 Conc Analysed No. of Samples CV 10 10 26. 4% 20 10 9. 4% 40 10 6. 9% 80 10 5. 5% 160 10 16. 9% Inter-assay coefficients of variation for sFlt-1 peptide standards at 30pg/ml and 15pg/ml added to 20 separate RIA's were: Mean Median CV 15pg/ml 13.8 14.0 11. 2% 30pg/ml 28.6 28.5 8. 7% 2. Digested Samples Intra-assay coefficient of variation from control serum pool and horse serum spiked with 30pg/ml NI-11 peptide: Control serum = 15. 3% (N=7) Horse + NI-11 = 10. 3% (N=7) Pooled pregnant samples were analysed individully or pooled prior to or after digestion Inter Assay CV - samples analysed individually = 5.1% (N=8) - samples pooled before digestion = 5.6% (N=8)

samples pooled after digestion before RIA analysis = 9. 1% (N=8) Assay Applications SFLT1 is clearly a key molecule in the regulation of circulating VEGF levels. However, its clinical application is currently being evaluated-in the past it has been limited by the lack of sensitivity and reproducibility of the ELISA. It has been shown (unpublished data) that sFlt-1 (total) concentrations are decreased early in gestation in patients who develop pre-eclampsia, which could imply that they would be more susceptible to the effects of VEGF. In addition, we have also shown that much greater circulating sFlt-1 concentrations exist than are currently measurable by ELISA and that complex sFlt-1 binding interactions exist in the circulation.

Within the cancer arena VEGF has been shown to play a major role in tumour angiogenesis. Thus, the present invention may be used to monitor tumour angiogenesis/activity through measurement of sFlt-1 levels in biological samples.

The invention is not limited to the embodiments hereinbefore described which may be varied without departing from the scope of the invention.