WO1990002759A1

WO1990002759A1 - Improvements relating to peptides

Info

Publication number: WO1990002759A1
Application number: PCT/GB1989/001084
Authority: WO
Inventors: Kenneth Bagshawe; Andrew Kardana; Brian COLES
Original assignee: Cancer Research Campaign Technology Limited
Priority date: 1988-09-14
Filing date: 1989-09-14
Publication date: 1990-03-22
Also published as: GB8821507D0

Abstract

A new peptide, urinary gonadotrophin peptide, (UGP) has been isolated from hCG from pregnancy urine. UGP dissociates into three fragments, two of which are new peptides while the third is substantially identical to the beta core fragment of hCG. UGP and antibodies to it are useful as diagnostics for various forms of neoplasia and pre-clinical neoplasia and are also useful as tumour markers and in antibody localisation.

Description

IMPROVEMENTS RELATING TO PEPTIDES

THIS INVENTION relates to peptides and is

particularly concerned with a new group of peptides that we have found to be associated with various cancer conditions.

Human chorionic gonadotrophin (hCG) is a glycoprotein hormone consisting of two dissimilar sub-units, alpha and beta which are joined together non-covalently. A fragment of beta hCG known as the beta-core fragment has also been identified.

We have now isola'ted and purified, from an hCG product prepared from pregnancy urine, a distinct peptide that we have designated urinary gonadotrophin peptide (UGP).

We have found that our UGP exhibits biological activity similar to hCG. Our further analysis of UGP shows this to be composed of three distinct peptides which we have designated fractions I, II and III. Fraction II has been shown, by us, to be identical with the beta core fragment of hCG. Fragments I and III, on the other hand, are quite different from the peptides found in pure hCG and we believe these to be new peptides.

We have used our UGP as an immunogen to raise antibodies. We have used UGP as an immunogen in rabbits to raise a polyclonal antibody that we have designated AK12 and we have used UGP as an immunogen in mice whose spleen cells were subsequently fused with myeloma cells to produce

hybridomas secreting monoclonal antibodies that we have designated 2C2 and 6D3. Both the polyclonal and monoclonal antibodies are able to recognise UGP and, more importantly, we have found that these antibodies recognise antigen associated with numerous neoplastic conditions. This indicates that UGP is of considerable importance as a marker of neoplastic conditions and the peptide and/or the

antibodies against the peptide are of interest for the localisation of anti-tumour compounds in the region of a tumour. In this way, both UGP and antibodies to it are of interest in the diagnosis and treatment of neoplastic conditions.

Accordingly, the present invention provides urinary gonadotrophin peptide in the form of an association of three peptides, fragments I, II and III where

Fragment II is substantially homologous to hCG betacore fragment; (by "substantially homologous" we mean that the sequence can differ from that of beta-core fragment by single amino acid substitution).

Fragment I is found to include the sequence 1-15:

Asp Val Lys X Asp Met Glu Val Ser Ser Pro Asp Gly Tyr Thr Fragment III includes the following amino acid sequence 1-15:

Lys Pro Gin Phe Thr X Ala Gin X Phe Glu Thr Gin Asn lle In the sequences for fragments I and III, X indicates an amino acid not yet positively identified.

The present invention includes both UGP in the form of a readily dissociable compound or a mixture of Fragments I, II and III in variable proportions.

Various methods are available for the preparation of UGP and the novel component peptides thereof. In our

preliminary experiments, we were able to isolate UGP from commercial preparations of hCG, itself obtainable from pregnancy urine. It is also possible to isolate UGP from pregnancy or non-pregnancy urine or to isolate it from human pituitary gland or to prepare it by synthetic or biosynthetic means.

Where the starting material is to be an hCG product, obtainable from urine, UGP can be isolated from hCG by a combination of chromatographic and electrophoretic

separations optionally together with affinity chromatography using an immobilised antibody that recognises UGP. An initial separation can be carried out with a molecular sieve and the various fractions obtained by this technique can then be subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) which separates the various proteins in the crude hGC still further. The UGP bands can then be identified and isolated utilising immobilised

antibody that recognises UGP.

UGP can also be isolated from human pituitary gland utilising a homogenate obtained with phosphate buffer and bovine serum albumin which can then again be subjected to separation by the molecular sieve and SDS-PAGE techniques mentioned above in association with the use of immobilised antibody.

Since the structures of significant regions of the component peptides of UGP have been largely determined, selected peptides or fragments thereof can be prepared by synthetic or semi-synthetic techniques in which the desired amino acid chain is built up by conventional peptide

synthesis, e.g. solid phase synthesis in which the peptide chain is built up from amino acid or peptide units as

required. In this way, it is possible to prepare sufficient of the peptide chains to be used as an immunogen for antibody construction, optionally after conjugation of the peptide with additional protein if the initial synthetic peptide is of insufficient molecular weight to be immunogenic in the host animal.

It is also possible, utilising the present knowledge of UGP, to produce it by genetic engineering techniques.

Such techniques involve the production of DNA encoding the desired amino acid sequence of the UGP fragment or fragments which can then be cloned, after linkage with appropriate control DNA fragments, in a suitable host cell, e.g.

bacterial, yeast or mammalian, so that expression of the desired polypeptide takes place. The necessary DNA encoding the desired UGP fragment can be prepared either synthetically or by isolation from a gene bank using probes encoding selected fragments from the novel sequences found in

Fractions I and III or by the isolation of mRNA from the pituitary homogenates mentioned above or from placental homogenates followed by conversion via cDNA into the required DNA for expression.

As mentioned above, the major applications of this invention are in the area of tumour marking and localisation of antibody in tumour regions. In order to put these aspects of the invention into effect, it is usually necessary to label the UGP of the invention and/or the antibodies to UGP of the invention. The exact nature of the label will be controlled by the specific application in question, for example, in a serological tumour marker assay, it would be useful to use radio-labels, particularly radio-active iodine such as ¹²⁵I. For other types of assay or histochemical applications, the label can be other materials that can be revealed by visual techniques, e.g. enzyme labels or

fluorescent labels and for immunoscintigraphy ¹³¹I, ¹¹¹Indium or technetium could be used.

When the antibodies of the invention are to be used as diagnostic tools, they will normally be used in assays such as radio-immuno assays or enzyme-linked immuno-absorbent assays and the antibodies will normally be present in

immobilised form. The antibodies can be immobilised onto any inert support for this purpose, usually an absorbent

cellulose based material or bonded onto a plastics material, e.g. polystyrene. The antibodies to be immobilised in this way may optionally be labelled and normally will be labelled with a radio-label if they are to be used in an RIA or with an enzyme label if they are to be used in an ELISA.

Using the RIA and IRMA techniques UGP has been found in normal adult urine at a low level. Patients with non- malignant diseases may have minor elevations in UGP excretion but generally fall within the normal adult range. Patients with a wide variety of cancers including lymphomas,

carcinomas of ovary, cervix, endometrium, breast, colon, stomach, pancreas, bladder etc., have been found to have increased levels of UGP in urine as have patients with all forms of trophoblastic tumours. In many cases the first manifestation of a cancer recurring after apparently complete surgical resection or suppression by radiation or

chemotherapy is an elevation of UGP in the urine.

The present invention extends to pharmaceutical compositions including the UGP or antibodies to UGP,

optionally labelled, including those formulated for

parenteral administration as well as test kits and components thereof for use in methods of diagnosis where at least one component of the test kit is UGP of the invention and/or antibody thereto, the UGP and/or antibody optionally being labelled. In accordance with still further aspects of the present invention, we provide a method of assaying body fluids, particularly urine, for UGP which comprises bringing the body fluid into contact with an antibody to UGP, the antibody being labelled and preferably immobilised. Assays of this nature are of particular value in routine screening of body fluids to give a preliminary indication of neoplasia or pre-clinical neoplasia and can give early warning which can be followed by more detailed localisation and/or imaging using antibodies to UGP in order to detect the site of the neoplasia more precisely.

Description of the drawings

Figure 1 shows graphically a molecular weight distribution of the three components of UGP.

Figure 2 shows a Western blot of a UGP fraction.

Figure 3 is a UV spectra showing the components of UGP.

Figure 4 is a Leydig Cell Bioassay of UGP and its components.

The following Examples are given to illustrate the invention.

(I) Sephadex G-100 Column Chromatoαrapy

The original source material for the isolation of UGP was a commercial preparation of hCG (Pregnyl, 5000U/ampoule by bioassay from Organon, Oss, Netherlands). This is derived from pooled collections of urines from pregnant women and is partially purified. 10 ampoules were used and these were dissolved in 0.05M phosphate buffer pH 7.5 (2ml). The solution was chromatographed on a column (85cm x 2.5cm) of Sephadex G-100 (Pharmacia, Uppsala, Sweden). Each fraction was assayed using an antibody to the beta-subunit (W14) and an antibody to the alpha-subunit (50/3 ) . The different peaks were concentrated using ultra filtration (YM-5 membrane, Amicon, Stonehouse, Gloucs, U.K.).

(2) Sodium Dodecyl Sulphate - Polvacrylamide Slab Gel

Electrophoresis (SDS - PAGE)

Both samples and appropriate molecular weight markers were reduced with mercaptoethanol and then separated by 10% PAGE (20% SDS). Protein bands were visualized with Coomassie Blue. Proteins from an identical gel were transferred to nitrocellulose paper using the "Western Blot" technique

(Burnette, 1981). The proteins were overlaid with either (i) a polyclonal rabbit antiserum (50/3) directed towards the alpha-subunit of hCG or (ii) a mouse monoclonal antiserum (W14) directed towards the beta-subunit of hCG; or (iii) a rabbit polyclonal antiserum (MW36) directed towards intact hCG (this had antibodies to both the alpha- and beta- subunits). The papers were then incubated with either ¹²⁵ι Concanavalin A and autoradiographed to show the glycoprotein bands containing either mannose or glucose residues.

(3) Immunopurification against immobilized antibodies to hCG Beta Subunit

Mouse monoclonal antibodies directed towards hCG beta-subunit (W14) were covalently linked to cyanogen bromide activated Sepharose CL/4B (Pharmacia). Samples were reacted with the immobilized antibodies and the bound antigen eluted with 3M ammonium thiocyanate and desalted immediately by use of a short column of Sephadex G-25 (Pharmacia).

(4) Production of antibodies to UGP

Antisera were raised in both rabbits and mice. Immunopurified UGP was covalently linked to thyroglobulin (1:1 by weight) using N-3-dimethyl aminopropyl carbodiimide hydrochloride as the linking agent.

The immunisation schedule for the rabbit (New Zealand

White) was as follows: a) A primer injection (subcutaneous) of 40ug UGP-conjugate in Freund's complete adjuvant (Sigma, Poole, Dorset, U.K.). b) Four weeks later a booster

injection (subcutaneous) was administered, also comprising 40ug in Freund's incomplete adjuvant. Blood was drawn two weeks later and then at fortnightly intervals to test for the production of antibodies AK12.

The immunisation schedule for the mice, for

monoclonal antibody production was as follows: a) Balb/c mice received an initial injection (subcutaneous) of 20ug of UGP- thyroglobulin conjugate in Freund's incomplete adjuvant, b) Three weeks later a booster injection (subcutaneous) of 10ug of UGP-conjugate in Freund's incomplete adjuvant was

administered, c) Three weeks after the first booster

injection, a second such injection (intra-peritoneal) was delivered, of 25ug UGP-conjugate in Freund's incomplete adjuvant. Three days after the final boost the mice were sacrificed and the spleen cells fused with the NS-1 myeloma cell line to form a hybridoma producing antibodies 2C2.

The "Pregnyl" material yielded three peaks as indicated in Fig.1.

(a) the major peak with an apparent molecular weight (m.wt.) of 70,000. This had both alpha- and beta-subunit activity and corresponded to the complete hCG molecule.

(b) a minor peak with an apparent molecular weight of 30,000. This had only alpha-sύbunit activity and is probably free alpha-subunit.

(c) another minor peak which had only beta-subunit activity with an apparent molecular weight of 15,000 (considerably less than whole beta-subunit). This was the UGP material.

The UGP was originally concentrated by ultrafiltration with a PM-10 membrane (Amicon). This membrane allows molecules of m.wt. less than 10,000 to pass through, it was found that UGP was in this category. Therefore a YM-5 membrane (m.wt. cut-off 5,000) was used so that the UGP was retained by ultra-filtration.

After SDS-PAGE (reducing conditions) the UGP material resolved into 3 bands when visualized by Coomassie Blue staining. Antibody overlay results showed only one band of activity. This band corresponded to a molecular weight of less than 10,000 and showed activity only with the antibodies to the beta-subunit or intact hCG. There was no binding with the antibodies to the alpha-subunit. (Fig.2)

Affinity chromatography of the "Pregnyl" -derived UGP produced only a small peak of unbound protein, as this starting material was already in a semi-purified form.

However this was not the case with the material extracted from the neoplastic patients urine which showed very large amounts of protein in the unbound fraction, with no UGP activity.

Extraction of UGP from Pituitary Gland

Approximately 1 gm (wet weight) of pituitary gland was homogenised with 3 mis phosphate buffer (0.05M, pH = 7.5) containing 0.1% (w/v) bovine serum albumin. The suspension was centrifuged for 10 mins (3500g) and the pellet discarded. The supernatant was chromatographed on a column containing Sephadex G-100 (Pharmacia, Uppsala, Sweden) and

immunopurified against the monoclonal antibody W14. UGP activity was detected using a 2C2/¹²⁵I-AK12 immunoradiometric assay (IRMA) system.

High Pressure Liguid Chromatography (HPLC)

Reverse phase HPLC was performed on a Gilson 714 system (Anachem, Luton, Beds., U.K.) using a Brownlee RP300 column (Anachem) 6.4mm x 50mm. The mobile phase was a linear gradient of 10% - 60% acetonitrile containing 0.05%

trifluoroacetic acid run over 30 minutes at a flow rate of 0.2ml/min. Approximately 10ug of sample was loaded and detection was monitored at 214nm.

Amino Acid Sequencing

50 - 100 pmol of sample was subjected to N-terminal amino acid analysis, using the Applied Biosystems 470A gas phase sequencer, with on line HPLC analysis utilising a 120A PTH analyser (Applied Biosystems, Warrington, U.K.).

Sequence homology with known peptides was analysed using the Beckman Micro-Genie software (Beckman, High

Wycombe, Bucks, U.K.). The various proportions of the fractions I, II and III present in UGP have been found to vary, depending upon the source of the UGP and reference is made to Figure 3 of the accompanying drawings which show ultra-violet spectra (absorbance units v. time) having peaks corresponding to each of the fragments.

Radioimmunoassay (RIA) for UGP

This procedure is a competitive binding assay, where labelled antigen and antibody directed against the antigen are incubated with sample. The addition of second antibody precipitates the antigen/antibody complex. The amount of bound tracer (labelled antigen) gives a measure of the amount of unlabelled antigen in the sample.

The antibody used in this assay was AK12 (rabbit polyclonal anti UGP) at a dilution of 1/5000 in dilution buffer (phosphate buffer pH 7.5 0.05M containing 0.1% bovine serum albumin) plus 1/400 normal rabbit serum. The standard curve was formed using 8 serial dilutions of UGP standard (800U/1). 100ul of the appropriate standard were incubated with 50ul of diluted antibody and 100ul of labelled UGP

(45000 cpm) overnight at room temperature. 50 ul of second antibody, goat anti-rabbit IgG (1/80) and 100ul of

polyethylene glycol 6000 solution (B.D.H. Chemicals ltd.,

Poole, Dorset, U.K.) were added and the mixture incubated for 2 hours at room temperature. The separation of bound from free tracer was achieved using a Kemtek 3000 automated RIA system (Kemble instruments Co. Ltd., Burgess Hill, Sussex, U.K.) by filtration through glass fibre filters (GF/F

Whatman, Maidstone, Kent, U.K.). The bound ¹²⁵I labelled UGP retained in the filter was quantified by the Kemtek 3000 and the data was processed by online microcomputer.

The lower limit of detection for UGP in this assay was 0.2ng/ml. The assay showed cross reactivity of 11% with hCG beta-subunit, 5% with hCG, 2.6% with LH and 0.4% with hCG alpha-subunit.

Immunoradiometric assay (IRMA) for UGP

This procedure is a typical "sandwich" IRMA, where a capture antibody is bound to a microtitre plate. The antigen is then incubated in the plate, followed by labelled second antibody directed against the antigen.

The wells of a microtitre plate were coated for 30 minutes at room temperature with 100ul of the monoclonal capture antibody 2C2 (40ug/ml bicarbonate buffer, 0.01M, pH9.5) directed against peptide 2 of UGP. The coating solution was then removed and the plate washed 3 times with distilled water. 100ul of blocking solution (2% BSA in dilution buffer: phosphate buffer, 0.05M, pH 7.5 containing 0.1% bovine serum albumin) was then incubated in each well for 30 minutes at room temperature. The solution was removed and the plate washed 3 times in distilled water. The

standard curve was made up of 9 serial dilutions of the UGP standard (100ng/ml, dilution buffer). Controls (UGP, 165 U/1 in dilution buffer) were also used. Urines were assayed diluted 1/2 in dilution buffer. The plate was then shaken at room temperature for 30 minutes, then incubated at room temperature overnight. The plate was washed 3 times in distilled water and 100ul of ¹²⁵ι labelled second antibody AK12 (rabbit polyclonal anti UGP) (25000 cpm) were then incubated in each well for 3 hours, shaking at room

temperature. The plate was then washed 3 times with

distilled water and the incorporated ¹²⁵ι determined using a gamma counter (1277 Gamma master, LKB, Bromma, Sweden). Data reduction and processing was carried out by an on line microcomputer, running Riacalc2 software (Pharmacia). The lower limit of detection for UGP in this assay was 0.4ng/ml UGP. The assay also showed cross reactivity of 2% with hCG beta-subunit and less than 1% with hCG, hCG alpha-subunit and LH. Biological Activity

Whereas beta-core fragment (peptide II) has been reported by others and confirmed by ourselves to be inactive in the rat Leydig cell testosterone assay for gonadotrophins, UGP has shown marked biological activity.

Determination of bioactivity of UGP

As part of an attempt to determine what role, if any, UGP plays in normal individuals it was decided to evaluate gonadotrophin like activity. The system used to quantify any bioactivity was the stimulation of testosterone production in murine Leydig cells in a modification of the assay by (Van Damme, M.P., Robertson, D.M., and Diczafalusy, E., (1974). An improved in vitro bioassay method for measuring

luteinizing hormone (LH) activity using mouse Leydig cell preparations). The quantity of testosterone produced was appraised by a testosterone/dihydrotestosterone

radioimmunoassay (RIA) kit (Amersham International pic,

Amersham, U.K.).

The cell suspension used in the bioassay was prepared as follows. The testes of 3 mice (CBA X Balb c) aged 2 to 3 months were, decapsulated and teased apart in a petri dish, containing RPMI 1640 medium (Northumbria Biologicals ltd, Cramlington, U.K.), with 2% foetal calf serum (FCS) (APP ltd, Brierley Hill, West Midlands, U.K.). The material was transferred to a specimen container and the volume made up to 50 ml with RPMI 1640 + 2% FCS. This preparation was stirred by magnetic bar for 15 minutes at room temperature. The cell suspension was filtered through a nylon mesh (pore size 100 x 100 μm). The filtrate was transferred to a 150 cm² tissue culture flask prior to incubation for 1 hour at 35ºC in a 5% CO₂ in air incubator. The cell suspension, after preincubation was centrifuged at 1200 rpm at room temperature for 10 minutes (MSE centuar2 centrifuge), then resuspended in 50ml RPMI + 2% FCS. The total cell count, assessed in a haemocytometer, was approximately 8 X 10⁵ cells/ml.

The materials assayed included human chorionic gonadotrophin (hCG), UGP, β core-fragment (BCF) and peptide III of UGP. β core-fragment is equivalent to peptide II of UGP. See figure 4.

From Fig. 4 it can be seen that a concentration of approximately 3ng/ml UGP induces the production of the same quantity of testosterone as approximately 11 μIU/ml hCG, ie. lmg/ml UGP is equivalent to 3.7 IU/ml hCG in this assay system. It can also be seen that β core-fragment (peptide II of UGP) is approximately 1300 times less potent and peptide III of UGP is approximately 600 times less potent than intact UGP in this system. The bioactivity shown by peptide III may be due to minor contamination by UGP as determined by HPLC, the same is suspected of the β core-fragment preparation. Immunohistochemical Distribution

Extensive studies have been made using polyclonal antibody AK12 and monoclonal antibody 2C2 to determine the distribution of UGP in normal and neoplastic tissue. The results are set out in Tables la, lb, II and III below.

Claims

1. UGP

2. An antibody to UGP.

3. A monoclonal antibody to UGP.

4. A diagnostic comprising a solid carrying UGP or an antibody thereto.

5. A diagnostic according to claim 4 carrying a revealing label.

6. A pharmaceutical composition comprising UGP or an antibody thereto together with a pharmaceutically

acceptable diluent or carrier, the UGP or the antibody optionally carrying a revealing label.

7. A method for the in vitro diagnosis of neoplasia or pre-clinical neoplasia which includes the step of

contacting a body sample from a patient to be diagnosed with UGP or antibody to UGP according to any one of the preceding claims.

8. UGP or antibody to UGP as defined in any one of claims 1 to 6 for use in a method of treatment on the human or animal body for therapy or in diagnosis.

9. A method of isolating UGP form hCG obtained from urine which comprises subjecting the hCG to chromatographic and electrophoretic purification including a step of treating a solution containing the UGP with an immobilised antibody to UGP.

10. A method of tumour marking or antibody

localisation in the region of a tumour which comprises introducting into a patient under investigation an antibody to UGP according to any one of claims 1 to 6, the antibody carrying a radio label.