Tashjian, A.M. et al, in Endocrinology, 1968, 82, 342-

52 describe the establishment of clonal strains of rat pituitary tumour cells that secrete growth hormone. In particular they describe the preparation of Growth Hormone

(GH)-secreting rat pituitary tumour cell line GH ₃ .

Until recently there had been no disclosure of a cell line derived from normal, i.e., non-cancerous, pituitary cells.

It has now been found that by infecting human pituitary cells with a temperature sensitive mutant of the early region T gene of SV40 (tsT), certain novel, stable human anterior pituitary cell lines are produced which have distinctive and advantageous properties.

According to one aspect of the invention there is provided a hormone and autocrine growth factor producing human pituitary cell line. According to a further aspect of the invention there are provided novel human pituitary cell lines which were deposited with European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, of Porton Down,

Salisbury, Wiltshire, SP4 OJG, United Kingdom, under deposit numbers 92112431 and 92112432 on 24th November 1992, under the Budapest Treaty and re-deposited under that Treaty with the same collection under deposit numbers 93052508 and 93052509 on 25th May 1993.

The CHP-2 cell line (deposit No. 92112431/93052508) when maintained at 39°C stains immunocytochemically for the common glycoprotein hormone subunit, alpha (which forms part of the intact pituitary hormones TSH (thyroid-stimulating hormone), LH (luteinizing hormone) FSH (follicle-stimulating hormone) and HCG (human chorionic gonadotrophin) ) and for the beta-subunits of TSH and HCG. Under similar conditions, the CHP-3 cell line (deposit No. 92112432/93052509) stains immunocyto- chemically for the above substances, but more weakly. Both cell lines are cytokeratin and chromogranin A positive, which indicates an epithelial origin. Both cell lines are positive for alpha subunit mRNA by in situ hybridisation. Solution and insitu hybridisation studies show that CHP2 cells contain detectable TSHβ, LHB, FSHβ, GH and PRL mRNA at 33°C, the levels of which are clearly accentuated at 39°C. However both cell lines appear to be negative for ACTH (adrenocorticotrophin), PRL (prolactin), GH and S-100 peptides.

As regards secretory activity, both cell lines have been found to secrete alpha subunit into the culture medium. This appears iπuπunologically to be identical to the natural product. Both cell lines secrete one or more autocrine growth-stimulating factors as assessed by the stimulating

effect of clone-conditioned medium on the growth of the same cell line, measured by 3H-thymidine incorporation into the cells, and by cell number estimations over periods of up to five days. Cell extracts of the cell lines also contain proliferative activity for GH ₃ cells as determined by a dose- dependent increase in [ ³ H]-thymidine incorporation and the accumulation of formazan dye in a colorimetric cell prolif¬ eration assay.

Both cell lines possess functional cell surface receptors for gonadotrophin-releasing hormone (GnRH) and cholinergic muscarinic agonists as assessed by the gener¬ ation of intracellular second messenger (inositol phos¬ phates). In addition, cells of CHP-2 (deposit No. 92112431/93052508) have functional receptors for thyrotrophin-releasing hormone (TRH).

According to another aspect of this invention there is provided a method of preparing a human pituitary cell line as described above which comprises preparing a culture of primary human pituitary cells, applying amphoteric retroviral tsSV40T vector to said cells and incubating said cells until foci of dividing cells develop.

In order to produce these human pituitary cell lines, normal human anterior pituitary tissue was removed three hours post mortem from a female patient aged 69 years who died following cardiac surgery. The tissue required enzymic dispersal with collagenase (lmg/ml), hyaluronidase (lmg/ml) and deoxyribonuclease (0.25mg mg/ml). Cells were maintained at 37°C in Dulbecco's modified Eagle medium (DMEM) contain-

ing 10% foetal calf serum (FCS).

An amphotropic retroviral vector encoding tsSV40 T was constructed as described by Wyllie et al in Cancer Res, 1992, 52 _. , 2938-2945. Supernatant from the ecotropic producer line psi2-tsA58-U19 (kindly donated by M. O'Hare, ICR, Sutton, United Kingdom) was used to infect the amphotropic packaging line psi-CRIP (Danos et al, proc. Natl Acad, Sci USA. 1988, 8_5, 6460-6464). These cells were maintained in DMEM containing 10% FCS and 400mg/l G418 (an antibiotic which is toxic for normal eukaryotic cells). The retroviral vector used also includes the neo bacterial gene, which confers resistance to G418. The resulting G418- resistant colonies were isolated and screened for retroviral production by determining titres for transduction of G418 resistance to murine NIH 3T3 fibroblasts and human A431 cells in colony-conditioned medium. The highest titre producer, designated psiCRIP-tsA58-U19-clone 2 was then used for the retroviral infection of primary pituitary cells.

Producer cells, on reaching approximately 90% conflu- ence, were incubated for twenty four hours in MCDB 211 (MCDB 211 is a mixture of 25% MCDB 104, 25% Hams F12 medium and 50% DMEM) containing 10% FCS (without G418), in order to generate retrovirus-containing medium. The medium was then filtered (0.45μm pore size), and applied to the primary human pituitary cell culture, which had been maintained in DMEM containing 10% FCS at 37°C for twenty four hours. The filtered retrovirus-containing medium was applied for two hours at 33°C together with polybrene 8μg/ml.

After washing the cells in MCDB 211 10% FCS (without G418), two days were allowed for proviral integration and expression, following which cells were maintained in MCDB 211 10% FCS containing G418 400mg/l in order to select for infected cells. The culture was maintained at 33°C, culture medium being replenished every 48-72 hours. Cells were inspected at regular intervals for the development of foci of dividing cells.

All media used contained antibiotics: benzyl penicillin (10 ⁵ U/1), streptomycin (lOOmg/1) and amphoterecin B (2.5mg/l).

Six weeks after retroviral infection, 6 foci of mitotically active, morphologically "epithelial" cells were observed. The two most active foci were isolated and passaged and designated CHP-2 (deposit No.92112431/93052508) and CHP-3 (deposit No. 92112432/93052509).

Both cell lines grow rapidly at 33°C in MCDB 211 with

10% FCS, with doubling times of 3 days (CHP-2) and 4 days

(CHP-3). At 39°C cell growth ceases and within 24 hours mitoses (which at 33°C are numerous) can no longer be observed.

The cells adhere well to the surfaces of culture flasks but can readily be removed by trypsinisation (0.05% trypsin in EBSS (Earles balanced salts solution) at 37°C for up to five minutes.

CHP-2 has undergone over 200, and CHP-3 over 150 cell divisions, with over 50 and 40 passages respectively.

The human pituitary cell lines of the present invention

may be useful for the transfection of human pituitary hormone genes, particularly glycoprotein hormones. The cell lines may also provide a suitable system for peptide production in particular for the production of glycoprotein hormones FSH, LH and TSH, also growth hormone and possibly insulin.

The autocrine growth factor produced by the cell lines may also be important in the growth of normal and adenomatous pituitary tissue. If so, the growth factor(s), its receptors and intracellular mitogenic path may provide targets for drugs acting to limit or even reverse tumour growth.

Thus the novel cell lines of the present invention could provide a useful laboratory model for developing drugs for the treatment of human pituitary tumours.

According to another aspect of the invention there is provided the use of a human pituitary cell line for the preparation of glycoprotein hormones.

According to yet another aspect of the invention there is provided the use of a human pituitary cell line for the preparation of autocrine growth factors.

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule l'bis)

For receiving Office use only For International Bureau use only tzT This sheet was received with the international application rπ This sheet was received by the International Bureau

Authorized officer Authorized officer ^-^

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13 ι«)

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule .3bis)

For receiving Office use oniy For International Bureau use only

| ■'-{ ^• 'T is sheet was received with the international application | j This sheet was received by the International Bureau

Authorized officer Authorized officer

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13Ws)

A. The indications made below relate to the microorganism referred to in the description on page 2 , ι l;i„n.e 1

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

EUROPEAN COLLECTION OF ANIMAL CELL CULTURES

Address of depositary institution (including postal code and country)

Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, SP4 OJG, United Kingdom.

Date of deposit 5yjϋ85SR5n Number

25th May 1 993 Deposit 93052509

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

SEE ADDITIONAL SHEET

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indicaύons are not for all designated Slate

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., 'Accessi Number of Deposit")

INDICATION IN RESPECT OF EUROPE

^■ f he applicant wishes that until the publication of the mention of the grant of a European patent or until the date on which the application is refused or withdrawn or is deemed to be withdrawn, ihe microorganism shall be made ava ⁱ lable as provided in Rule 28(3) of the Implementing Regulations under the European Patent Convention only by the issue of a sample to an expert nominated by the requester (Rule 28(4) of the said Implementing Regulat ⁱ ons), the applicant must inform, by a written statement, the International Bureau accordingly before complet ⁱ on of technical preparations for publication of the international application.

INDICATION IN RESPECT OF AUSTRALIA

Aft applicant m*? givtf notice that the furnishing of a sample of a microorganism shall only be effected prior to ϊ?π ^g ? ^ ³ P ^aten fv° ^{r pπ0r} . ^{t0 the ,apsin} 8' ^{refusal or w} «^drawal of the application, to a person who is a sk ⁱ lled addressee w ⁱ thout an interest in the invention (Regulation 3.25(3) of the Australia Patents Regu _¬ lat ⁱ ons). A not ⁱ ce to this effect must be filed by the applicant with the Australian Patent Office before the

INDICATION IN RESPECT OF NORWAY

The applicant 1-ffξpτequesfthal, until the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act.

INDICATION IN RESPECT OF FINLAND

The applicant la-a? requestfthat, until the application has been laid open to public inspection (by the National Board of Patents and Registration), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, ihe furnishing of a sample shall only be effected lo an expert in the art.