AU612766B2 - Monoclonal antibodies - Google Patents

Description

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AUSTRALIA

Patents Act 612766 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: 0 .0 .00.

0 3 3 3 *3 3 *3 3* Name(s) of Applicant(s): Address(es) of Applicant(s): Actual Inventor(s): APPLICANT'S REF.: CAP of PI 6257 BUNGE (AUSTRALIA) PTY. LTD.

6th Floor 616 St. Kilda Road, Melbourne, Victoria Australia RODNEY JOHN FIDDES Address for Service is: PHILLIPS, ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled: MONOCLONAL ANTIBODIES The following statement is a full description of this invention, including the best method of performing it known to applicant(s): P19/3/84 A primed mouse was killed by cervical dislocation and the spleen aseptically removed. Splenocytes were The present invention relates to monoclonal antibodies, a method for the preparation thereof and use thereof.

Follicle-stimulating hormone (FSH) or follitropin, is a glycoprotein hormone secreted by the pituitary gland.

FSH, and other pituitary glycoprotein hormones (Luteinizing hormone LH, thryoid stimulating hormone TSH,), consist of an o and 3subunit. The o(-subunit of these hormones is common within a species whilst the 6-subunit differs and confers the specificity of each hormone.

FSH has a role in sexual function in both male and female animals. In males, it seems the most important function of FSH is in the maturation and nutrition of sperm, whilst in females FSH has a role in development of the ovum and ovulation. Exogenous FSH can be used for superovulation in embryo transfer programs, or for ovulation increases to I give greater fecu-dity. However variation in the response of treated animals can be a major problem and may be attributed S* to batch variation and contaminents present in FSH preparations, particularly the presence of another pituitary glycoprotein hormone, LH (Luteinizing hormone)..

The major methods known in the prior art of ovine FSH (oFSH) extraction include for the production of crude extracts of oFSH from lamb pituitary glands by the use of ethanol and acid precipitation (Sherwood, et al (1970) J. Biol Chem.

245: 2329-2336), and (ii) ammonium sulphate precipitation (Reichert, L.E. in Methods in Enzymology Vol. XXXVII Ed. O'Malley B.W.

and Hardman J.G. Academic Press Inc. N.Y. 1975).

Depending on the method used unwanted proteins are precipitated out by the stepwise addition of cold ethanol or ammonium sulphate. A final addition of ethanol or ammonium sulphate precipitates the active component as well as some contaminants. These methods produce crude FSH extracts containing 0.5 1.0 u/mg of crude protein.

Further purification steps using the crude pituitary extracts include gel filtration and ion-exchange chromatograpy (Sherwood.,\ucts otaie rane in p y from P, SLIAV \T .Ri.Cied Aruccwts \oVie e raocngeb o-0 r.

The purified products obtained range in potency from 2 2a 110 to 188 units of oFSH per mg of total protein.

However these prodecures incur large expense, are time consuming and produce only small amounts from each pituitary gland, and as a result large quantities of FSH are rarely available for veterinary use, especially FSH of a consistent potency.

Accordingly it is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties related to the prior art.

Accordingly in a first aspect, the present invention provides a method of purifying ovine follicle stimulating hormone; said method including the steps of: providing: a source of impure ovine follicle stimulating hormone; and, (ii) an affinity matrix including therein Smonoclonal antibodies specific for ovine follicle stimulating hormone; o oo. applying the source of impure ovine follicle stimulating hormone to the affinity matrix in a manner such that ovine follicle stimulating hormone therein can become bound to the monoclonal antibodies in the matrix which are specific for the follicle stimulating hormone; ooooo allowing the ovine follicle stimulating hormone to bind with the monoclonal antibodies, in the matrix, which are specific therefor; and S• eluting the bound ovine follicle stimulating hormone, as a purified ovine follicle stimulating hormone, from the affinity matrix; wherein the monoclonal antibodies specific for ovine follicle stimulating hormone are formed from cell lines of the group consisting of: BFR87-70, BFR87-124 or a functionally equivalent cell line.

The monoclonal antibody against follicle stimulating hormone (FSH) may be produced by: T- 2b providing: B cells which produce antibodies against follicle stimulating hormone; and, myeloma cells; (ii) fusing the B cells with the myeloma cells to form hybridoma; (iii) propagating the hybridoma; and, (iv) collecting antibody produced by the hybridoma.

2 0 i o 1- f- 110 Lu 188 unltLs o -rSH-perg -fetal However these prodecures incur large expense, ar time consuming and produce only small amounts from ach pituitary gland, and as a result large quantities of SH are rarely available for veterinary use, especially SH of a consistent potency.

Accordingly it is an object of the resent invention to overcome, or at least alleviate, e or more of the difficulties related to the prior art.

Accordingly in a first asect, the present invention provides a process for prod cing a monoclonal antibody against follicle stimulati" hormone (FSH), which process includes providing a cell capable of producing antibodies against FSH, and a yeloma cell, fusing he B cell with the myeloma cell, and pro agating a hybridoma formed thereby.

he process for producing a monoclonal antibody accor ng to this aspect of the present invention may further in ude harvesting an antibody produced by said hybridoma.

The B cell capable of producing antibodies against FSH may be selected from spleen cells and lymph node cells of a test animal. The test animal may be a mouse, rat or the S• like. The B cells may be obtained from an animal immunised with FSH or an immunogenic fragment thereof. A BALB/c mouse is the preferred animal for immunisation. Once-primed or hyper-immunised animals may be used as the source of antibody .producing lymphocytes or splenocytes. In a particularly preferred form, hyperimmunised mouse spleen cells are used to make the fused cell hybrids.

The myeloma cell may be of any suitable type.

Specialized myeloma cell lines which have been developed from lymphocyte tumours for use in hybridoma-producing fusion procedures are known in the prior art.

Several myeloma cell lines may be used for the production of fused cell hybrids, including P3/X63-Ag 8, NS-I type myeloma cells, e.g. P3/NSI/l-Ag 4-1, Sp2/0-Agl4 and S194/5.XXO.BU.l. In the example of the present invention, a SNS-l-type myeloma cell (derived from BALB/c mice) is the 3 cC-L preferred cell line.

The fusion step according to this aspect of the present invention for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells includes mixing somatic cells with myeloma cells. The proportion may vary from approximately 20:1 to 1:1, preferably 10:1, respectively. The fusion step may be carried out in the presence of an agent or agents that promote the fusion of cell membranes. It is preferred that the same species of animal serve as the source of the somatic and myeloma cells used in the fusion procedure. The fusion-promoting agents used may include Sendai virus or polyethylene glycol (PEG).

Because fusion procedures produce viable hybrids at very low frequency when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly 5 every 2 x 10 spleen cells), it is preferred to have a 'means of selecting the fused cell hybrids from the remaining 9: unfused cells, particularly the unfused myeloma cells. A means of Ocetecting the desired antibody-producing hybridomas among othe- resulting fused cell hybrids is also necessary.

Generally, the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the myeloma cells which normally would go on dividing indefinitely.

These cells are selected against in hypoxanthine/aminopterin/ thymidine (HAT) medium, a medium in which the fused cell hybrids survive due to the HPRT-positive genotype of the spleen cells. The use of myeloma cells with different genetic deficiencies other enzyme deficiencies, drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.

The use of highly discriminatory screening assays during the cloning and selection of hybridoma cell lines assists in selecting for monoclonal antibodies having the desired specificity.

The detection of antibody-producing hybrids can be achieved by any one of several standard assay methods, including enzyme-linked immunoassay and radioimmunoissay 4 techniques. Immunohistology can also be used for antibody detection and selection. The detection method preferred is a double-antibody radioimmunoassay employing radioactive-iodine labelled oFSH. Other assay techniques which have been tested may lead to false positive results.

Once the desired fused cell hybrids have been selected and cloned into individual antibody-producing cell lines, each cell line may be propagated in either of two standard ways. A sample of the hybridoma can be injected into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumours secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can be tapped to provide monoclonal antibodies Sin high concentration. Alternatively, the individual cell S lines may be propagated in vitro in laboratory culture vessels; the culture medium, also containing high concentrations of a single specific monoclonal antibody, can be harvested by decantation, filtration or centrifugation.

Accordingly, in a further a-spcct of th- prcsjt invention there is provided a continuous ne which produces a monoclonal antibody- follicle stimulating hormone including a idoma formed by fusing a B cell Scapable oducing antibodies against follicle stimulating =or-nO with.a my nm rFp11.

Preferably, the hybridoma cell line is formed by fusing an NS-1 myeloma cell with a spleen B cell obtained from a BALB/c mouse immunised with follicle stimulating hormone. An ovine follicle stimulating hormone (oFSH) preparation may be used to immunise the mouse.

The mouse is immunised with FSH extracted from lamb or pig pituitary glands combined with an adjuvant. The hybridoma cell line grows in tissue culture using a nutrient solution. The cell line when analysed for purity after repeated tissue culture passage by dilution cloning will exhibit antibody producing FSH specific clones at a frequency of 100%.

Hybridomas of interest may be dilution cloned to ensure the selection of a single cell line. Hybridomas may 1 1--11- l ~L11-. l- 1 be dilution cloned at least four times. Particular examples of hybridoma cell lines are those designated BFR87-70 and BFR87-124 which are raised against ovine FSH.

Samples of the above-mentioned are maintained in the Bunge (Australia) Pty. Ltd. Cell Collection at 89 Flemington Road, North Melbourne, Australia. The invention described herein is not limited in scope by the hybridoma cell lines exemplified since these embodiments are intended as illustrations of one aspect of the invention and any equivalent cell lines which produce a functionally equivalent monoclonal antibody are within the scope of the invention.

Samples of the following hybridoma cell lines have been deposited with the European Collection of Animal Cell Cultures (ECACC) Culture Deposit No.

SBFR87-70 89010301 BFR87-124 89010302 In 1~ 1 fEr- aspect of the present invention there is provided a monoclonal antibody against follicle stimulating hormone produced from a continuous cell line which produces a monoclonal antibody against follicle stimulating hormone including a hybridoma formed by fusing a B cell capable of producing antibodies against follicle stimulating hormone with a myeloma cell.

":The monoclonal antibody may be produced from a k hybridoma cell line as described above. Specific examples of monoclonal antibodies include those formed by hybridoma cell lines BFR87-70 and BFR 87-124 which are raised against ovine FSH, samples of which are maintained in the Cell Collection facility of Bunge (Australia) Pty. Ltd. at 89 Flemington Road, North Melbourne, Australia.

I-n-a-f-u-r-t-her---a-pec-t---f-t-he-p-r-eet--i-nvei-tn tbhore is provided a method of purifying follicle :1mulating hormone (FSH) which method includes providing a source of im u-rFSH, and a mono nal antibody against FSH bound to a matrix; and sing the impure FSH through the monoclonal a nrly Matrix i-n rapture FRH_ The method of purification may furthse- include k- 6 i i;:i eluting the captured FSH from the monoclonal antibody matrix.

The monoclonal antibodies bound to the matrix may be any of the monoclonal antibodies described above.

A simple method for the purification of follicle stimulating hormone is of importance since those methods known in the prior art are both costly and time consuming, and, result in a product of low purity. A purified FSH is of importance as it provides a greatly improved biological response in-vivo. For example, cintamination with LH decreases the effects of administered FSH. High purity FSH is extremely desirable for application in human medicine.

The development of technology enabling the purification of FSH through monoclonal antibody affinity chromatography has provided a process whereby the supply of commercial quantities of a highly purified FSH product is possible.

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i Highly purified FSH has some distinct advantages over the impure FSH products currently available. Batch 5: variation of commercial FSH is extensive and the quality of the in-vivo response is poor by comparison. FSH purified from impure batches of FSH using the presently described technology has little if any batch variation and provides a greatly improved response in-vivo. The improved response is evident in all species tested.

•m Accordingly, in a still further aspect of the present invention there is provided a purified follicle stimulating hormone (FSH). Preferably the purified FSH is a S purified ovine FSH.

The matrix used may, be of any suitable type. A chromatographic gel is preferred. An affinity chromatographic gel may be used. The gel sold under the trade designation AFFIGEL 10 and available from Bio-Rad Laboratories has been found to be suitable. AFFIPREP 10 and (Bio-rad Laboratories) are also suitable.

The step of passing the impure FSH through the monoclonal antibody matrix may be undertaken in any suitable container. An affinity column may be used for relatively small scale work. For large scale work, a large container or vat may be used. The large container or vat may be rotated, agitated or stirred to ensure complete contact between the 7 I iI impure FSH and the monoclonal antibody matrix. A rotor bottle may be used as a suitable container.

The captured FSH maybe eluted from the monoclonal antibody matrix utilising any suitable elution solution. An organic acid solution may be used. A pH buffered solution may be used. A citrate/citric acid buffer may be used to remove unbound material from the column.

Stepwise elutions with 0.1M citrate/citric acid solutions with a pH of approximately 6.75 to 2.0 have been found to be suitable.

The in-vivo response to FSH may be improved through the application of the following new technologies High purity FSH, and (ii) Monoclonal antibodies to FSH.

In a further aspect of the present invention there is provided a method of inducing oestrus and/or increasing fecundity in female animals which method includes administering to the animal an effective amount of a purified follicle stimulating hormone. Anoestrus animals are thereby induced to cycle and ovulate.

The term animals as used herein in the description and claims includes domestic animals including sheep, goats, cattle, pigs and the like as well as other animals including humans.

For sheep and goats the ovine follicle stimulating hormone may be administered in amounts of from approximately 2 to 10 iu. For cattle the follicle stimulating hormone may be administered in amounts of from approximately 2 to 20 iu.

In a further preferred aspect of the present invention there is provided a method of substantially increasing the number of ova produced during a single oestrus cycle in a female animal (superovulation). This method includes administering to the animal an effective amount of a purified follicle stimulating hormone. The number of ova produced may be increased to a level of at least about preferably 15-20.

For sheep and goats the follicle stimulating hormone may be administered in amounts of from approximately 4 to iu.

For cattle the follicle stimulating hormone may be 8 1. administered in amounts of from approximately 4 to 20 iu.

It has now been discovered that monoclonal antibod.es to follicle stimulating hormone may block and/or enhance the biological activity of follicle stimulating hormone in animals.

Control of FSH bioactivity in vivo using monoclonal antibodies is advangeous when using FSH for superovulation, (ii) increasing fecundity, and (iii) manipulation of the oestrus cycle.

Accordingly, the methods described above may further include administering simultaneously with, or after, FSH administration to the animal approximately 0.25 to 3.0 mg of an purified monoclonal antibody against FSH formed by a hybridoma cell line.

The advantages already described for superovulation are also applied to the low dose usage of FSH for increasing Sfecundity, i.e. twinning and manipulation of the oestrus *cycles. By using these monoclonal antibodies with low doses of FSH the incidence of twin births in a variety of species can be better controlled. Twinning is therefor easier and more repeatable, and the numbers of unwanted triple and high S. multiple births are reduced.

Accordingly in a further aspect of the present Sinvention there is provided a method of increasing ovulation to provide twinning in female animals which method includes administering to the animal an effective amount of a purified a S follicle stimulating hormone.

Preferably the animal to be treated is a sheep, goat or cow and the amount of follicle stimulating hormone administered is in the range of approximately 2 to 20 iu.

More preferably the method of increasing ovulation to provide twinning further includes administering simultaneously with or after follicle stimulating hormone administration to the animal approximately 0.50 to 1.0 mg of a purified monoclonal antibody against follicle stimulating hormone formed by a hybridoma cell line.

In a preferred aspect of the present invention the method of inducing oestrus and/or increasing fecundity further includes administering to the animal to be treated an 9 10 effective amount of a monoclonal antibody against follicle stimulating hormone (formed by a hybridoma cell line) simultaneously with or after follicle stimulating hormone administration.

The monoclonal antibody may be any of the antibodies described above. The monoclonal antibody may be administered in any suitable effective amount. For sheep and goats the monoclonal antibody may be administered in amounts from approximately 0.25 to 1.0 mg. For cattle the monoclonal antibody may be administered in amounts from approximately to 3.0 mg. For pigs the monoclonal antibody may be administered in amounts from appro'imately 0.2 to 0.5 mg.

In the method of super ovulation, the monoclonal antibody may be administered for sheep and goats in amounts of from approximately 0.5 to 2.0 mg. For cattle the monoclonal antibody may be administered in amounts of from approximately 0.5 to 3.0 mg.

When administering FSH monoclonal antibodies for *control of FSH in-vivo, an accurate assessment of the 20 required dose is preferred.

The antibody dose suitable is dependent on several variables including: the monoclonal antibody selected, (ii) FSH dose, (iii) time following FSH injection of antibody injection, (iv) species in which it is being used, size of the animal, and (vi) number of times the animal has been previously treated The use of monoclonal antibodies for controlling FSH in vivo whether for use in superovulation, increasing fecundity i.e. twinning or for induction of oestrus as described for ewes and cows has similar application in goats, humans, dogs, cats, pigs and other mammals.

The amount of antibody necessary to be effective will vary with the particular antibody used, the species of animal, the level of FSH treatment and time of administration \NLI~ following FSH injection. For example, in a preferred form, 4+ 4 where from approximately 15 to 20 iu of FSH is injected into -W cJC I a ewe, we have found that approximately 0.05 to 0.50 milligrams of antibody may be used aopending on the variables already described. The monoclonal a.itibody treatment may be undertaken contemporaneously with the FSH treatment or may be undertaken after FSH treatment.

Monoclonal antibodies administered simultaneously or after FSH administration can modify the biological activity of FSH. This modification may reduce or enhance the activity of the FSH. Both enhancement and reduction of FSH bioactivity can be incorporated in the same regime. It is possible to achieve a 5 10 times increase in potency of FSH with "FSH-antibody" complex.

Preincubation, simultaneous injection or independent administration of FSH and the monoclonal antibody can enhance the activity of the FSH via the formation of antibody-FSH complexes, resulting in greater biological activity.

Equally so the formation of a different antibody-FSH complex can effectively reduce the bioactivity of the FSH or previously complexed FSH by nullifying the biological activity of the FSH.

In a still further aspect of the present invention there is provided a method for the detection of follicle stimulating hormone (FSH) in a mammal. The detection assay may be an immunofluorescent assay, radioimmuno-assay, enzyme-linked immuno assay or agglutination assay. For the IgM immunoglobulin class monoclonal antibodies an agglutination assay is preferred. A haemagglutination assay is preferred.

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99*99 In a particularly preferred form the detection assay is a haemagglutination inhibition assay.

The sample to be tested may be of any suitable type. Sera, plasma, saliva or urine samples or extracts thereof may be used.

It would be understood that the presence of FSH may then be detected by visual means. Where FSH is present in the serum of interest, the FSH will bind to available binding sites on the monoclonal antibodies. When the FSH coated mammalian red blood cells are added there is effectively no antibody to bind to the antigen on the cells and there is a resultant inhibition of agglutination. Non-agglutinated 11 w 12 cells subsequently slide around the round bottom of the solid support well to form a dark dot of cells.

The solid support may be of any suitable type. A microtitre plate or tray or test tube may be used. A microtitre plate or tray of the round bottom type is preferred. The monoclonal antibodies may be introduced as a solution. A phosphate buffered saline solution may be used.

In a further aspect the present invention provides a veterinary or pharmaceutical composition for the modification of the biological activity of FSH in mammals including an effective amount of a monoclonal antibody selected from those described above or mixtures thereof in a unit dosage for.

The veterinary or pharmaceutical composition may further include a veterinarily acceptable or pharmaceutically acceptable diluent carrier or excipient therefor. The S monoclonal antibody may be provided in solution. A buffered saline solution may be used. A phosphate buffered solution may be used.

a The present invention will now be more fully described '2 20 with reference to the accompanying examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

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EXAMPLES

M4ATERIALS AND METHODS Manufacture of Monoclonal Antibody to ESH BALB/c mice were injected IP with 5 mg of a purified preparation of oFSH or pFSH (50 u/mg, ammonium sulphate precipitated according to tche method of Reichert 1975 followed by several ion exchange chromatography steps, in ul of PBS emulsified in a ratio of 1:1 with Freund's Complete Adjuvant (ECA) This immunfication step was repeated four t ime s at weekly intervals. One week a f te r the final injection, mouse sera, obtained by tail bleeding were examined for FSH reactivity and specif-icity by radio-immunoassay (RIA). Mice having the highest serum titres were boosted four days prior to fusion with 100, ug of the same purified preparation of oFSH or pFSH (50 u/mg) in 0.1 ml of PBS by tail-vein injection.

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A primed mouse was killed by cervical dislocation and the spleen aseptically removed. Splenocytes were obtained by teasing of the spleen in 10ml GKN (0.8 g NaC1, 0.4 g KC1, 1.42 g Na 2

HPO

4 2H10, 2.0 g D-glucose and 0 01 g Phenol Rod in 1 litre of double distilled water).

Solution: Cells were collected by centrifugation. NS-1 myeloma cells (2 x 107) P3/NS1/1-Ag4-1, grown in tissue culture media (Dulbecco's Modified Eagles Medium, DME and sodium bicarbonate 0.2% w/v) were collected by centrifugation (200 g for 5 min. at room temperature) and resuspended in ml GKN solution.

The splenocytes and NS-1 myeloma cells were mixed in a 50ml centrifuge tube (Falcon) and centrifuged at 70g for minutes at room temperature. The supernatant was then poured off to leave a pellet of cells. These cells were resuspended to form a slurry, by gently tapping the tube, in a minimum of residual supernatant. 1.5ml of sterile 50% polyethyleneglycol at 37 C [PEG, Art. 9727 Polyethyleneglycol 4000, Merck] prepared by autoclaving 1.0g PEG with 1.0 ml of double distilled water in a sealed container, was then added to the slurry over 90 seconds, with gentle mixing. After a further seconds of mixing, GKN at 37°C was carefully added over 2 minutes followed by a further 8 ml over 3 minutes. Finally 30ml GKN was then gently added down the side of the centrifuge tube.

The cell suspension was immediately centrifuged at room temperature for 3 minutes at 70g and the supernatant poured off. The tube was tapped gently to form a slurry of cells before resuspension in 100 ml of HAT selective media (tissue culture media containing 13.6 mg hypoxanthine, 0.19 mg aminopterin and 3.87 mg thymidine per litre of media).

Samples of the cell suspension (0.5 ml) were then added to each well of eight 24-well flat bottom Linbro plates (Flow 6 Laboratories) each containing 1.5 x 10 rat thymocytes in HAT selective media. The thymocytes were prepared on the day prior to the fusion and incubated overnight at 370C in a humidified 5% CO 2 atmosphere.

Thirteen days after fusion, supernatants were screened by RIA and Immunohistology. Selected wells were dilution cloned in 96-well flat bottom Linbro plates with 13 1 I thymocytes in HAT selective media.

Radioimmunoassay ELISA (enzyme-linked immunosorbent assay) systems have often been used as a method of detecting monoclonal antibodies in hybridoma supernatants. However, in such a saystem antigen is presented in a solid phase, i.e. adherent to the plastic ELISA plate, and many scientists have reported false positive results (Miller, Goldsby, and Bolt, D.J. J. Endocr. (1987), 115: 283-288). To test for antibody which is required for use in liquid-phase applications, i.e. affinity chromatography and injection of antibody into animals as a suppressor of FSH activity, a liquid-phase assay was developed and is described below.

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i i I_ i;i Radioimmunoassay Screening Procedure Hybridomas secreting antibodies to ovine FSH were initially detected using a double-antibody RIA. A pure preparation of oFSH (75 u/mg from NIH, Bethesda, United States of America) is radioiodinated using lodogen (Pierce, United States of America). In this procedure 5 ug of oFSH is incubated with 0.5 mg of 125I (Amersham IMS 30) and 50 ul of 0.1M Phosphate buffer, pH 7.4) in the presence of 1.2 mg of Iodogen coated onto a glass tube. After incubation for minutes at room temperature the radioiodinated oFSH 125 125 I-oFSH) is separated from free I by chromatography on a 10 ml G-25 column.

Hybridoma supernatants (100 ul) are added to a polystyrene test tube (3DT tube, Johns) with 100 ul of 0.1% BSA/PBS containing approximately 100,000 counts per minute 125 (cpm) of 125 I=oFSH. After incubation for2 hours at room .temperature 100 ul of a precipitating second antibody is Sadded. The second antibody is an antimouse antibody made in rabbits (RAM, affinity purified Silenus Laboratories) conjugated to Sepharose 4B (CnBr- Sepharose 4B, Pharmacia).

After vortexing tubes are left at room temperature for 1 hour Sand 1 ml of cold phosphate buffer is added. Tubes are immediately spun at 3,000 rpm for 15 min. at 4 C. The 125 non-antibody bound I-oFSH in the supernatant is aspirated and the pellet containing the 125 I-oFSH-monoclonal antibody to oFSH 2nd antibody complex is counted in a gamma spectro-photometer.

Immunohistology SFresh tissues (pituitary glands) were collected from the local abattoir. The tissues were fixed in Bouins solution for two hours and then formalin overnight. After tri nAg, tissues were embedded in paraffin by processing through four 1 hour changes of absolute ethanol at 4C four changes of xylene at room temperature, and four changes of liquified paraffin at 56°C, and then left to solidify overnight at Tissue sections of 6 um were cut using a microtome (Leitz) and floated onto glass slides from a warm 1% gelatin water bath. Slides were then incubated overnight at 15 I- 1 37 C to adhere the sections. Prior to use, sections were rehydrated by 5 minute incubations in each of the following solutions; xylene, fresh xylene, absolute ethanol, fresh absolute ethanol, 70% ethanol and running water.

Neat hybridoma supernatants were placed directly onto the sections and the slides incubated in a humidified box for 2-3 hours at room temperature. After gentle rinsing with PBS, sections were covered with rabbit anti mouse IgG conjugated to horse radish peroxidase (RAM-HRP, Dakopatts, Denmark) diluted 1:75 in PBS containing 2% normal sheel serum and incubated for a further one hour. After rinsing the slides were submerged in a substrate solution containing 0.14g 3,3'-diaminobenzamidine tetrachloride in 250ml 0.01M citrate buffer, pH 5, plus 250ul of 30% hydrogen peroxide.

Within 10 minutes the reaction was stopped by rinsing the slides in distilled water.

Sections were counterstained with Harris' haematoxylin and lithium carbonate, and then dehydrated by one minute rinses through 70% alcohol, absolute alcohol, fresh absolute alcohol, xylene and fresh xylene for mounting. Supernatants were considered positive for FSH antibody !if pituitary cell cytoplasm stained in the Scharacteristic way described in the literature.

Results Ovine FSH The fusions performed on the spleen cells from mice immunized against oFSH yielded 2 hybridomas which produce antibodies to oFSH (see Table 1).

TABLE 1 RIA Histology BFR87-70 30% BFR87-124 24% a. max. binding of total 125I-oFSH added b. is staining of cytoplasm of pituitary cells Improvement of Biological Activity An improvement of the biological activity of FSH preparations can be achieved by affinity chromatography using monoclonal antibodies to FSH. There is also evidence of an increased potency of injected FSH when the FSH is coupled to the monoclonal antibodies.

16 r 7- L ~I _i 16a The extraction of purified, biologically active FSH from crude extracts by affinity chromatography confirms the specificity of the antibody and allows accurate assessment of the biological properties of FSH.

Selection of Monoclonal Antibody suitable for Affinity Chromatography To select a monoclonal antibody suitable for Affinity Chromatography purification of ovine follicle stimulating hormone, mice were immunized with a purified form of ovine follicle stimulating hormone (60 u/mg). This ovine follicle stimulating hormone was purified using traditional gel and ion-exchange chromatography. While not an absolutely pure preparation of follicle stimulating hormone, immunization using this preparation immunized the production of antibodies to non-follicle stimulating hormone proteins.

A liquid-phase assay was developed to screen hybridoma supernatants. This assay allowed the selection of suitable antibodies at the earliest possible stage and eliminated the S chance of false positives often found using solid-phase 20 assays as described by Miller Bolt, D.J. and Goldsby S (1983), J. Immunological Methods, 59:277-280. Two hybridoma cell lines BFR87-70 and BFR87-124 were found to secrete antibodies which bound radiolabelled ovine follicle stimulating hormone (NIH-oFSH-I-1) in the liquid-phase assay. These monoclonal antibodies were also tested immediately for their specificity by immunohistological examination. Both monoclonal antibodies BFR87-70 and BFR87-124 showed positive staining of the basophil cells of the sheep anterior pituitary, the only known source of ovine 30 follicle stimulating hormone, but were completely negative against all cells of the sheep posterior pituitary, liver, heart, kidney, brain and muscles.

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from crude extracts by affinity chrgoa py confirms the specificity of the anibody and allows accurate assessment of Affinity ChromatoaraD hv ES- Affinity Chromatoaraohv Approximately 200mg of each monoclonal antibody is purified (from cell culture or ascites fluid) and coupled to Affigel 10 at 16mg/ml of gel-. The columns are prepared for each affinity gel preparation, with a small portion (200 ul) of gel for use in establishing optimal binding and eluting conditions. Optimal conditions were investigated by incubating 10 ul of gel in 200 ul of buffers at various pH values. After 1 hour at room temperature, the test tubes are 125 centrifuged and any non-bound 1I-oFSH aspirated. The gel 125 containing any I-oFSH is then counted in a gamma spectrophotometer.

OPTIMAL BINDING FOR BFR87-70 Buffer 0.1 Ethanolamine-HCl pH PBS pH 0.1M Phosphate pH 0.1M Citrate/Citric Acid pH 0.1M Citrate/Citric Acid pH S 0.1M Citrate/Citric Acid pH 0.1M Citrate/Citric Acid pH 0.1M Citrate/Citric Acid pH 0.1M Citrate/Citric Acid pH 0.1M Citrate/Citric Acid pH 11.0 7.4 7.0 6.0 5.5 5.0 3.5 3.0 Counts Bound 2,610 68,920 64,490 58,710 54,240 53,270 38,460 19.550 3,820 3,490 Counts Added 2 47 44 37 36 26 13 2.6 2.4 *se For optimal elution, a series of tubes were 125 incubated with 10 ul gel plus 1I-oFSH diluted in PBS.

125 These tubes withbound 1I-oFSH, were further incubated with buffers at various pH values, spun and counted.

17 18 OPTIMAL ELUTION FOR BFR87-70 Buffer 0.1 Ethanolamine-HCl

PBS

0.1M Phosphate 0.1M Citrate/Citric 0.1M Citrate/Citric 0.1M Citrate/Citric 0.1M Citrate/Citric 0.1M Citrate/Citric 0.1M Citrate/Citric 0.1M Citrate/Citric Acid Acid Acid Acid Acid Acid Acid pH 11.0 pH 7.4 pH 7.0 pH 6.0 pH 5.5 pH 5.0 pH 4.5 pH 4.0 pH 3.5 pH 3.0 Counts Bound Counts Eluted 8,270 91 90,780 0 86,560 6 85,180 8 80,120 8 77,010 21 35,800 46 27,380 67 12,160 87 9,160 91

V*

Si t Relative to counts bound in first incubation with PBS.

Optimal binding occurs using PBS as the binding buffer while maximum elution from the gel matrix, of 125I-oFSH, is achieved with 0.1M citratP/citric acid pH 3.0 buffer. This is preferred to the pH 11.0 buffer as FSH is less susceptible to damage at pH This protocol was tested in a 2ml column, which indicated that this procedure was suitable (Table 2).

Affinity Column (2 ml) 125 125 I-oFSH added 3,047,800 Counts washed through 840,000 (28%) Counts bound 2,200,000 72% Counts eluted with pH citrate/citric acid 2,116,550 96% of bound counts Large scale trials can be undertaken by either column or batch methods. The affinity-purified FSH can be tested for biological activity using the hCG augmentation assay for FSH of Steelman and Pohley (Endocrinology, 53:604-616, 1953) or the porcine testes receptor assay of Maughium-Rogister et al, (Eur. J. Biochem. 86:121-131, 1978).

This purified preparation of FSH will allow an exact dose of FSH to be given. Batch variations found in crude preparations and the problems caused will be alleviated.

These antibodies to ovine and porcine FSH will also allow the investigation of enhancement of FSH activity by the coupling of biological FSH and its antibody and injecting this complex into the object. This treatment may decrease the dosage rate of FSH substantially and hence decrease the T- J.

':i 19 i i cost of FSH treatment since the major cost of the treatment is the purified FSH.

Further Evidence that the Monoclonal Antibody BFR87-70 is specific for only ovine FSH Further evidence that the monoclonal antibody BFR87-70 is specific for only ovine follicle stimulating hormone was established by purifying ovine follicle stimulating hormone using the monoclonal antibody BFR87-70 by the following procedure: Pituitary glands from lambs are homogenized in distilled water at 4.8 litres of water per kilogram of pituitaries and the pH adjusted to 5.5. This homogenized preparation is stirred overnight (18-20 hours) at 4 0

C.

The supernatant of the homogenized pituitaries is collected by centrifugation at 8,000 x g for 1 hr. This supernatant is brought to pH 7.4 and the Affinity Matrix is S then added. The affinity matrix is Monoclonal Antibody BFR87-70 coupled to Affiprep (Biorad, USA) at 9 mg of S antibody per mg of Affiprep.

0 After mixing for 16 hrs at 4 0 C, the affinity matrix is collected and poured into a chromatography column connected to an LKB Wallac fraction collector and a U.V.

spectraphotometer to detect protein peaks.

The affinity matrix column is washed with 0.lm S phosphate buffer pH 7.4.

The affinity matrix column is then washed with .lm phosphate buffer pH 7.4 plus 0.5m NaC1 until the elute S is free of protein.

Antibody-bound protein is then eluted in 0.lm citrate/phosphate buffer pH 3.0 and collected into 0.lm Tris S pH 7.4. These elution conditions were found to be optional for elution of all antibody-bound protein.

The eluted protein is then dialyzed against PBS (phosphate buffered saline) and evaluated for ovine follicle stimulating hormone activity by RRA (radio-receptor assay), Steelman-Pohley assay and PAGE (poly-acrylamide gel electrophoresis).

I

I

19a *9* 9 Biological Potency of ovine FSH Ovine FSH purified by affinity chromatography using the monoclonal antibody BFR87-70 from whole ovine pituitary glands appears as a single bond on SDS-PAGE (line 5, Figure The biological potency of this ovine FSH was assayed in the Steelman-Pohley Bioassay (Steelman, S.L. and Pohley, F.M. Endocr. (1953) 53:604-616) and shown to have a potency of 200-220 units/mg relative to the NIH-oFSH-Sl standard reference. The highest biological potency of ovine FSH cited in the literature is 188 u/mg (Sherwood, Grimek McShan, 1970, J. Biol. Chem. 245, 2328-2336) following purification by gel filtration and ion-exchange chromatography. Other works (Papkoff and Ekblad, 1970, Biochem. Biophys. Res. Commun. 40, 614-621) purified ovine FSH to 37 u/mg and Grimek and McShan (1974, J. Biol. Chem.

249, 5725-5732) to 133 u/mg relative to the NIH reference S standard NIH-oFSH-Sl. All these preparations prepared by conventional biochemical techniques have less biological S activity per mg of protein than the ovine FSH prepared by 20 affinity chromatography using the monoclonal antibody BFR87-70.

Degree of Purification by Immunoaffinity Chromatography Miller, Goldsby and Bolt (1987, J. Endocr. 115, 283-288) have purified bovine FSH by affinity chromatography and shown that bovine FSH from bovine pituitaries can be enriched by a factor of 1500 fold.

Ovine FSH purified from ovine pituitaries by affinity S chromatography using the monoclonal antibody BFR87-70 showed a purification factor of 9500 fold (Table This is a 6 fold better enrichment than that obtained by Miller, Goldsby and Bolt (1987) with bovine FSH.

99.4i 9* *4 ,u~ur,,rn l :I 19b TABLE 2 The amount of protein and activity of ovine FSH from 1 kq. of ovine pituitaries at various stages of purification by affinity chromatography using the monoclonal antibody BFR87-70

FRACTION

20 Supernatant from 1 kg. of ovine pituitaries Affinity Column Washings (non-retained)

TOTAL

PROTEIN (mg) 128,401 128,000 Total FSH Activity by RRA (relative to NIH-oFSH-Sl) 2,877 206 *Specific Activity u/mg 0.022 0.002 Affinity Column, Eluted at pH3 (sample retained) 11.9 2,495 209.7 Specific Activity defined as units of FSH per mg of protein relative to NIH-oFSH-S1 reference standard.

Purification Factor of 9,500

C

Ii:

I

r Y 19c Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

0 >4 4, 7J

Claims (5)

1. 3. A method according to claim 1 wherein said step of providing an affinity matrix including therein monoclonal antibodies specific for ovine follicle stimulating hormones includes a step of: providing monoclonal antibody against ovine follicle stimulating hormones by: PkI~~2 CO A 1ii 21 providing: B cells which produce antibodies against follicle stimulating hormone; and, myeloma cells; (ii) fusing the B cells with the myeloma cells to form hybridoma; (iii) propagating the hybridoma; and, (iv) collecting antibody produced by the hybridoma.
2. 4. A method according to claim 3 wherein: said step of providing B cells comprises obtaining B cells from an animal immunized with ovine follicle stimulating hormone; and, said step of providing myeloma cells includes providing NS-1 myeloma cell line. A method according to claim 3 or claim 4 wherein said step of fusing the B cells with the myeloma cells includes mixing somatic cells with the myeloma cells in the presence of a fusion promoting agent. S6. A method according to claim 5 wherein the fusion promoting agent includes material selected from the group consisting of: Sendai virus and polyethiyene glycol.
3. 7. A method according to any one of claims 1 to 6 wherein: said step of providing B cells includes providing spleen B cells obtained from a BALB/c mouse immunized with ovine follicle stimulating hormone: and, said step of providing myeloma cells includes providing NS-1 myeloma cells.
4. 8. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples.
5. 9. A purified ovine follicle stimulating hormone when prepared by the method of any one of claims 1 to 8. DATED: 6 March 1991 PHILLIPS ORMONDE FITZPATRICK Attorneys For: BUNGE (AUSTRALIA) PTY LTD 0^a J 04 pLA *9419h) 9 i :L