WO2012136519A1

WO2012136519A1 - Use of neutralizing prolactin receptor antibodies for the treatment and prevention of antiestrogen-resistant breast cancer

Info

Publication number: WO2012136519A1
Application number: PCT/EP2012/055433
Authority: WO
Inventors: Christiane Otto; Christoph Freiberg
Original assignee: Bayer Pharma Aktiengesellschaft
Priority date: 2011-04-06
Filing date: 2012-03-27
Publication date: 2012-10-11

Abstract

The use, respectively the method of use of neutralizing Prolactin receptor antibodies (PRLR antibodies) for the treatment and prevention of antiestrogen-resistant breast cancer is described. These antibodies can be combined with one ore more of several other cytotoxic agents, when used in treatment and prevention of antiestrogen- resistant breast cancer.

Description

Use of neutralizing prolactin receptor antibodies for the treatment and prevention of antiestrogen-resistant breast cancer

The present invention refers to the use, respectively the method of use of neutralizing Prolactin receptor antibodies for the treatment and prevention of antiestrogen-resistant breast cancer.

Prolactin (PRL) is a polypeptide hormone composed of 199 amino acids. PRL belongs to the growth hormone (GH), placental lactogen (PL) family of polypeptide hormones and is synthesized in lactotroph cells of the pituitary and in several extrapituitary tissues such as lymphocytes, mammary epithelial cells, the myometrium, and the prostate. Two different promoters regulate pituitary and extrapituitary PRL synthesis (BioEssays 28: 1051 -1055, 2006).

PRL binds to the PRL receptor (PRLR), a single transmembrane receptor belonging to the class 1 cytokine receptor superfamily (Endocrine Reviews 19:225-268, 1998). The PRLR exists in three different isoforms, the short, the long, and the intermediate form that can be distinguished by the length of their cytoplasmic tails. Upon ligand binding, a sequential process leads to PRLR activation. PRL interacts via its binding site 1 with one PRLR molecule and then attracts via its binding site 2 a second receptor molecule leading to an active dimer of PRLRs. PRLR dimerization leads to the predominant activation of the JAK/STAT (Janus Kinase/Signal transducers and activators of transcription) pathway. Upon receptor dimerization, JAKs (predominantly JAK2) associated with the receptor, transphosphorylate and activate each other. In addition the PRLR is also phosphorylated and can bind to SH2-domain containing proteins such as STATs. Receptor bound STATs are subsequently phosphorylated, dissociate from the receptor and translocate to the nucleus where they stimulate transcription of target genes. In addition, activation of the Ras-Raf-MAPK pathway and activation of the cytoplasmic src kinase by PRLRs have been described (for review Endocrine Reviews 19:225-268, 1998).

PRLR-mediated signalling plays a role in a variety of processes such as mammary gland development, lactation, reproduction, mammary and prostate tumor growth, autoimmune diseases, general growth and metabolism, and immunomodulation (Endocrine Reviews 19:225-268, 1998; Annu. Rev. Physiol. 64:47-67,2002). It has been suggested that the PRLR is overexpressed in human breast cancer (J Clin Endocrinol Metab 83:667-674, 1998) and that negative interference with the PRL- PRLR interaction might have positive impact on breast cancer treatment (Int. J.

Cancer: 55(5): 712-721 , 1993). There is evidence, that in general, plasma prolactin levels correlate with breast cancer risk (Cancer Lett 243: 160-169,2006). In patients with tamoxifen-resistant breast cancer, the development of progressive disease correlates with prolactin plasma levels (Eur J Surg Oncol 20: 1 18-121 , 1994).

Transgenic mice overexpressing prolactin are more prone to the development of breast cancer (J Clin Invest 100:2744-2751 , 1997).

Currently, there is no medication available that enables complete interference with PRLR-mediated signalling. The only way to interfere with PRLR-mediated signalling based on current medications is the inhibition of pituitary PRL secretion by use of bromocriptine and other dopamine receptor 2 agonists (Nature Clinical Practice Endocrinology and Metabolism 2(10): 571 -581 , 2006). These agents however, do not suppress extrapituitary PRL synthesis that can compensate successfully for the inhibition of pituitary PRL synthesis leading to almost unimpaired PRLR-mediated signalling (Endocrine Reviews 19:225-268, 1998). Therefore it is not surprising that dopamine type 2 receptor agonists were not beneficial in patients suffering from breast cancer or autoimmune diseases such as systemic lupus or rheumatoid arthritis (Breast Cancer Res. Treat. 14:289-29, 1989; Lupus 7:414-419, 1998) although prolactin has been implicated in these diseases. Local prolactin synthesis in breast cancer cells or lymphocytes which plays a pivotal role in mammary carcinoma or autoimmune diseases, respectively, was not blocked by dopamine receptor agonists.

Therefore, prolactin receptor antagonists that completely interfere with prolactin mediated signalling irrespective whether the involved prolactin was synthesized locally or in the pituitary, represent a novel treatment paradigm for breast cancer that will be superior if compared to dopamine 2 receptor agonists that only block pituitary prolactin synthesis.

Antibodies binding to PRLR for breast cancer diagnosis were claimed in

WO03/004989 for the first time. In said WO03/004989 numerous genes and corresponding proteins including PRLR being overexpressed in several breast tumor tissues were regarded as suitable markers for early detection of malignancy. In WO03/008583, focussing on lymphoma and leukemia, carcinoma-associated genes and proteins are described including PRLR. In addition, agents neutralizing the activity of the carcinoma-associated proteins are described. Furthermore, in WO2006/1 10585 PRLR as a cancer marker for breast cancer and other cancer types (lung, prostate and skin cancer) are described. This time, monoclonal antibodies with antagonistic activity were claimed based on two

experimental evidences: a) blocking of the PRLR expression by PRLR-specific siRNA in breast cancer MCF7 cells led to inhibition of proliferation of these cells and b) prolactin induces STAT5 and MAPK phosphorylation in breast cancer cells such as T47D. However, neither any evidence is provided that a monoclonal antibody indeed inhibited proliferation of breast cancer cell lines nor any concrete antibody is disclosed.

In contrast, the first in vivo proof of concept that PRLR antagonists successfully interfere with breast cancer development has been published in 1988.

In the DMBA mouse breast tumor model, a monoclonal PRLR antibody led to reduced incidence of mammary tumors (Am J Pathol 133:589-595, 1988).

First PRLR antibodies, claimed for the prevention and treatment of breast cancer, are described in US 2007/0269438. The expression products (antibodies) from five hybridoma cell lines are described by their ability to tightly bind breast cancer cell lines T47D and MCF7 and to block PRL-mediated signalling (STAT5-, MAPK-, AKT- phosphorylation). It is described that three of the studied antibodies are blocking T47D cell proliferation. No in vivo proof of concept has been disclosed. Moreover, no evidence beyond the state-of-the-art knowledge has been provided for the statement that especially after the patient's cancer become antiestrogen-independent, the anti- PRLR antibody may be administered to the patient.

A second set of monoclonal PRLR-specific antibodies together with disclosed primary sequences is described in WO 2008/022295. Although these antibodies were claimed as therapeutic agents for breast, lung and prostate cancer, a lymphoma model served to show the antiproliferative effect of these agents in vivo. On the other hand, peptidergic PRLR antagonists that are derived from prolactin and that carry an N-terminal deletion and a point mutation (e.g. delta1 -9G129R prolactin) also behave as antagonists of the PRLR, however they suffer from reduced half-life time (15-20 min) and low potency if compared to prolactin (Pituitary 6:89-95,2003). Therefore, neutralising PRLR antibodies are superior especially with regard to the inhibition of enhanced autocrine prolactin signalling that plays a role in breast cancer and prostate cancer.

In conclusion, many data have been published indicating that PRLR might play a causative role in breast cancer. And conceptually, potent PRLR antagonists seem the appropriate agents to demonstrate the therapeutic value of PRLR inhibition for breast cancer. Nevertheless, no in vivo proof of concept has been provided up to now that PRLR blockade indeed leads to inhibition of tumor growth (except lymphoma) and especially of anti-estrogen resistant breast tumor growth.

Antiestrogen-resistant breast cancer

Worldwide, breast cancer contributes to 24% of all cancer cases (J Steroid Biochem Molec Biol 42:21 1 -221 , 1992). Approximately 75% of breast cancer patients suffer from tumors that are positive for the estrogen receptor (ER) and progesterone receptor (PR) and these patients are subjected to antiestrogen therapy (Cancer 1 13:2385-

2397,2008). Basically, there are four classes of compounds used in ER positive breast cancer that interfere negatively with ER-mediated signalling:

a) tamoxifen acts as a selective estrogen receptor modulator (SERM) with

antagonistic activity at the ER in the mammary gland and thus blocks ER- mediated signalling in response to estradiol

b) aromatase inhibitors such as anastrozole and letrozole inhibit the synthesis of estradiol

c) antiestrogens such as fulvestrant block ER-mediated signalling in response to estradiol or growth factor activation and lead to rapid degradation of the ER. d) GnRH analogues such as buserelin and goserelin and GnRH antagonists such as cetrorelix inhibit the hypothalamic-pituitary-gonadal axis and

cause a drop in estradiol levels While almost all ER negative breast cancers exhibit intrinsic antiestrogen resistance, approximately 50% of ER positive breast cancer patients fail to respond to

antiestrogenic therapy either from the very beginning (intrinsic antiestrogen resistance) or after prolonged time of antiestrogenic treatment (= aquired antiestrogen resistance) (J Steroid Biochem Molec Biol 42:21 1 -221 , 1992). Finally, antiestrogen resistance provokes recurrence and progression of breast cancer. Therefore, alternative treatment paradigms for antiestrogen-resistant breast tumors are urgently needed.

Currently the following hypotheses exist to explain resistance towards treatment with compounds that negatively interfere with ER-mediated signalling:

The estrogen receptor can be activated by agonists such as estradiol or in a ligand-independent manner by several growth factor pathways such as the EGFR (epidermal growth factor receptor), the human epidermal growth factor receptor 2 (Her-2/neu), and mitogen-activated protein kinase (MAPK). Growth factor activation of the ER leads to serine 1 18 phosphorylation of the ER and its activation in the absence of the ligand estradiol (Cancer 1 13;2385-2397, 2008). Enhanced growth factor signalling is observed in breast cancer cells that are resistant against the approaches a), b), and d) mentioned above that rely on the antagonization of estradiol signalling or on the suppression of estradiol levels (Cancer 1 13;2385-2397, 2008).

Absence of estrogen receptors in tumor cells or expression of mutant ERs in tumor cells.

3. Altered expression of ER coregulator proteins that increase the agonistic

effects of tamoxifen that normally acts as an ER antagonist in the breast.

Faslodex is an antiestrogen that leads to degradation of the ER protein and thus prevents growth factor-mediated activation of the ER. Therefore, faslodex is often used in cases of tamoxifen resistance. However, breast cancer cells can even develop resistance against faslodex (Endocrine-related cancer 12:S29-S36, 2005). Faslodex resistance is often due to enhanced EGFR signalling, i.e. survival and proliferation of the breast cancer cells becomes completely independent of the ER.

According to this observation, a promising treatment paradigm in antiestrogen- resistant breast cancer is the blockade of growth factor signalling. EGFR inhibitors such as gefitinib as well as herceptin, a blocker of Her2/neu signalling or AG1024, an inhibitor of the IGF-IR (insulin growth factor I receptor) are used either alone or in combination with faslodex to treat antiestrogen-resistant breast cancer. In general, these treatment paradigms prolong the disease-free interval compared to

antiestrogen-only therapy, but in the end, resistance develops also against these treatments. Therefore, there is still a need for new alternative treatment paradigms in case of antiestrogen resistant breast tumors.

The antibodies of the pharmaceutical composition of the present invention interfere with PRLR-mediated signaling stimulated by pituitary- and breast cancer cell-produced prolactin and represent an additional option for the treatment of antiestrogen-resistant breast cancer. We employed these antibodies in the SHN mouse model that has been shown to develop hyperplastic alveolar nodules (HANs) and breast cancer

spontaneously and in an accelerated manner after pituitary transplantation, i.e.

hyperprolactinemia (Laboratory animal Science 37;200-202, 1987; Eur J Cancer Clin Oncol 21 ; 1 109-1 1 1 1 , 1985).

In this mouse model antiestrogenic compounds that are clinically used have also been analysed, such as faslodex (ICI) and cetrorelix (GnRH antagonist). Surprisingly, antiestrogenic approaches did not prevent the establishment of hyperplastic alveolar nodules (HANs) that represent preneoplastic mammary gland growth and later give rise to breast cancer, whereas PRLR antibodies completely abolished the generation of HANs (Example 1 , Figure 1 ). In other words the SHN mouse mammary tumor model represents an in vivo model for breast cancer that is resistant towards treatment with compounds that negatively interfere with ER signalling and thus resembles a model for antiestrogen-resistant breast cancer. PRLR antibodies show full efficacy in this model of antiestrogen-resistant breast cancer in the preventive (Example 1 , Figure 1 ) and therapeutic setting (Example 2, Figure 2). It should be noted that antiestrogen-resistant breast cancer constitutes a major health problem. Around 50% of estrogen receptor positive breast cancers show intrinsic or acquired resistance to standard treatment paradigms (J Steroid Biochem Molec Biol 42:21 1 -221 , 1992). Basically, new therapies for antiestrogen-resistant breast cancer 5 are steadily required, since after a while patients become also resistant towards

treatment paradigms that might be efficient in antiestrogen-resistant breast cancer for a certain period of time.

Currently no antibodies are available for the treatment or prevention of antiestrogenic⁾ resistant breast cancer that show sufficient results, respectively have sufficient

properties.

It is therefore an object of the present invention to solve that problem by PRLR antibodies that prevent and treat antiestrogen-resistant breast cancer based on an 15 unexpected result from an in vivo antiestrogen-resistant tumor model.

PRLR-antibodies which are herewith incorporated by reference are described in PCT/EP2010/067740, PCT/EP2010/067742, PCT/EP2010/067743,

PCT/EP2010/067744, PCT/EP2010/067746 and PCT/EP2010/067747. However, 0 these references are silent with respect to any use in antiestrogen-resistant breast cancer.

It has now been found that said antibodies are surprisingly specific to and have a high affinity for PRLR and this way neutralize the PRLR-mediated signalling and that can deliver a therapeutic benefit to the subject.

5 For example, antibodies that can surprisingly be used for prevention and treatment of antiestrogen-resistant breast cancer and which are described in PCT/EP2010/067740, PCT/EP2010/067742, PCT/EP2010/067743, PCT/EP2010/067744,

PCT/EP2010/067746 and PCT/EP2010/067747, are more potent and thus offer the opportunity of an improved side-effect profile, i.e. reduced risk of immunogenicity due

30 to reduced dosages, in contrast to those antibodies as disclosed in WO 2008/022295.

Now, PRLR-antibodies that are disclosed in PCT/EP2010/067740,

PCT/EP2010/067742, PCT/EP2010/067743, PCT/EP2010/067744, PCT/EP2010/067746 and PCT/EP2010/067747 and that can surprisingly be used as medicament for prevention and treatment of antiestrogen-resistant breast cancer are described in the following table 1 .

Antibodies as disclosed in WO 2008/022295 and that are of interest for study, are listed in table 2.

To show superiority of the instant antibodies over the known antibodies from

WO 2008/022295 in antiestrogen-resistant breast cancer, the instant antibodies have been compared with those antibodies from WO 2008/022295 (see Example 3)

Table 1: Sequences of the antibodies

The instant antibodies are specific to and have a high affinity for PRLR and this way neutralize the PRLR-mediated signaling and that can deliver a therapeutic benefit to the subject. Blockade of PRLR activation by neutralizing PRLR antibodies leads to a complete inhibition of PRLR-mediated signaling. In contrast, dopamine receptor agonists can only interfere with enhanced PRLR-mediated signaling in response to elevated pituitary prolactin secretion, but not with enhanced PRLR-mediated signaling due to an activating PRLR mutation or due to locally elevated prolactin production.

It has now been found that the instant antibodies can be used for the prevention and therapy of antiestrogen-resistant breast cancer and show furthermore surprising properties with regard to the state of the art antibodies. The instant antibodies may be administered as the sole pharmaceutical agent or in combination with one or more additional therapeutic agents where the combination causes no unacceptable adverse effects. This combination therapy includes administration of a single pharmaceutical dosage formulation which contains an instant antibody and one or more additional therapeutic agents, as well as

administration of the antibody and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, an instant antibody and a therapeutic agent may be administered to the patient together in a single iv. or oral dosage composition such as a solution, tablet or capsule, or each agent may be administered in separate dosage formulations. In particular, the compounds of the present invention may be used in fixed or separate combination with other cytotoxic agents such as alkylating agents, anti-metabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti- tumor drugs. In this regard, the following is a non-limiting list of examples of secondary agents that may be used in combination with the compounds of the present invention: AB-1001 , ABI-007, Abirateronacetat, Acitretin, Aclarubicin, Acolbifen, Actimmun, Affinitak, Afinitor, Aflibercept, Alaninat, Aldesleukin, Alendronsaure, Alfaferon,

Aldesleukin, AZD2171 , Alemtuzumab, Alitretinoin, Allopurinol, Aloprim, Aloxi,

Altretamin, Amifostin, 9-Aminocamptothecin, Aminoglutethimid , Aminopterin,

Amonafid, Ampligen, Amrubicin, Amsacrin, Anastrozol, Androcur, Android,

Andromustin, Angiostatin, Anzmet, Apaziquon, Aplidine, Apocyproteron, Apoflutamid, Aranesp, Arglabin, Asentar, Arsentrioxid, Aromasin, Arzoxifen, Asoprisnil, L- Asparaginase, Atacicept, Atamestan, Atrasentan, Avastin, Axitinib, 5-Azacytidin, Azathioprin , BAM-002, BCG oder tice-BCG, Belinostat, Belotecan, Bendamustin, Bestatin, Betamethason-Acetat, Betamethason-Natriumphosphat, Bevacizumab, Bexaroten, Bicalutamid, Bleomycin, Bleomycin-Sulfat, Bosentan, Bosutinib,

Brostallicin, Bortezomib, Brivanib, Broxuridin, Busulfan , Calcitonin, Calcitriol,

Campath, Camptothecin, Canfosfamid, Capecitabin, Carboplatin Carfilzomib,

Carmofur, Carmustin, Casodex, Cetuximab, Catumaxomab, CCI-779, CDC-501 , Cefeson, Celebrex, Celmoleukin, Cerubidin, Cetuximab, Chlorambucil,

Chlormadinonacetat, Cilengtide, Cisplatin, Cladribin, Clodronsaure, Clofarabin, Colaspase, Combretastatin, Corixa (melanoma vaccine), Crisnatol, Cyclophosphamid, Cytarabin , Cyproteron-Acetat, Cytosinarabinosid, Dacarbazin , Daclizumab,

Dactinomycin , Darinaparsin, Dasatinib, DAST, Daunorubicin, DaunoXome, Decadron, Decadron-Phosphat, Decitabin, Delestrogen, Denileukin Diftitox, Depomedrol,

Deslorelin, Dexrazoxan, Diethylstilbestrol , Diflomotecan, Diflucan, 2,2'- Difluordeoxycytidin, Dinatriumpremetrexed, DN-101 , Docetaxel , Doxercalciferol, Doxifluridin, Doxorubicin, Doxorubicin-MTC, Dronabinol, Dutasterid, dSLIM, DW- 166HC, E7820, Edotecarin, Eflornithin, Eligard, Elitek, Ellence, Emend, Endostatin, Enocitabin, Epimbicin, Epristerid, Epirubicin, Epoetin-alfa, Epogen, Epothilon und seine Derivate, Epratuzumab, Eptaplatin, Ergamisol, Erlotinib, Estrace, Estradiol, Estramustin-Natriumphosphat, Ethynylcytidin, Ethinylestradiol, Ethyol, Etidronsaure, Etopophos, Etoposid , Everolimus, Exemestan, Exatecan, Exisulind, Fadrozol,

Farston, Fenretinid, Filgrastim, Finasterid, Flavopiridol, Filgrastim, Floxuridin,

Fluconazol, Fludarabin, Fludarabin-Phosphat, 5-Fluordeoxyuridin, 5-Fluordeoxyuridin- Monophosphat, 5-Fluoruracil (5-FU), Fluoxymesteron, Flutamid, Folfox. Folfiri.

Formestan, Fosteabin, Fotemustin, Fulvestrant, Galliumnitrat, Gammagard, Gefitinib, Gemcitabin, Gemtuzumab, Gimatecan, Gleevec, Gliadel, Gossypol, Glufosfamid, Goserelin, Granisetron, Granisetron-Hydrochlorid, Halofuginone, Hesperadin,

Hexamethylmelamin, Histamin-Dihydrochlorid, Histrelin, Histrelin-Hydrogel-lmplant, Holmium-166-DOTMP, Hycamtin, Hydrocorton, 10-Hydroxycamptothecin,

Hydroxycarbamid, Hydroxyharnstoff, Hydroxyprogesteron-Caproat, erythro- Hydroxynonyladenin, Hydroxyharnstoff, Ibandronsaure, Ibritumomab, Idarubicin , Ifosfamid, Imatinib, Imatinibmesylat, Imiquimod, Interferon, Interferon-alpha, lnterferon-alpha-2, lnterferon-alpha-2a, lnterferon-alpha-2p, lnterferon-alpha-n1 , lnterferon-alpha-n3, Interferon-beta, Interferon-gamma, lnterferon-gamma-1 a,

Interferon gamma-n1 , lnterleukin-2, Intron A, Intron-PEG, Ipilimumab, Iressa,

Irinotecan, Ixabepilon, JSM 6425, Keyhole Limpet-Hamocyanin, Krestin, Kytril, L- 651582, L19-IL2, L-Asparaginase, Lanreotid, Lapatinib, Lasofoxifen, LBH589,

Lenograstim, Lentinan, Lentinan-Sulfat, Letrozol, Leucovorin, Leuprolid, Leuprolid- Acetat, Levamisol, Levofolinsaure-Calciumsalz, Levothroid, Levoxyl, Libra, Lobaplatin, Lomustin, Lonafarnib, Lonidamin, Lumiliximab, Lupron, Lurtotecan, Mafosfamid, Mapatumumab, Marinol, Masitinib, Mechlorethamin, Mecobalamin,

Medroxyprogesteron-Acetat, Megestrol-Acetat, Melphalan, Menest, 6-Mercaptopurin, 6-Mercaptopurinribosid, Mesna, Methotrexat, Metvix, Mifepriston, Miltefosin,

Minocyclin, Minodronat, Miproxifen Mitolactol, Mitomycin C, Mitotan, Mitumomab, Mitoxantron, MK-2206, Modrenal, Molgramostim, MS-209, MS275, liposomales MTP- PE, MX-6, Myocet, Nafarelin, Nandrolondecanoat, Nedaplatin, Nelarabin, Neulasta, Nemorubicin, Neovastat, Neumega, Neupogen, Nilotibib, Nilutamid, Nimustin,

Nolatrexed, Nolvadex, Nolatrexed, NSC-631570, Obatoclax, Oblimersen, OCT-43, Ocfosfat, Ocfosfit, Octreotid, Ondansetron-Hydrochlorid, Onko-TCS, Orapred,

Oregovomab, Osidem, Oxaliplatin, Ozogamicin, Paclitaxel, Paclitaxel-Polyglutamat, Pamidronat-Dinatrium, Palladia, Panitumumab, Panobinostat, Patupilone, Pazopanib, Pediapred, Pegaspargase, Pegasys, Palonosetronhydrochlorid, Pemetrexed,

Pemtumomab, Pentostatin, Pelitrexol, Pertuzumab, /V-Phosphonoacetyl-L-aspartat (PALA), Picibanil, Pilocarpin-Hydrochlorid, Pirambicin, Pirarubicin, Pixantron,

Plicamycin, PN-401 , Porfimer-Natrium, Prednimustin, Prednisolon, Prednison,

Premarin, Procarbazin, Procrit, ProMune, Prost-aid, Provenge, QS-21 , Quazepam, Raloxifen, Raltitrexed, Ranibizumab, Ranimustin, Ranpirnas, Rapamycin, R-1549, RDEA1 19, Rebif, Rebimastat, Recentin, Regorafenib, Removab, 13-c/s-Retinsaure, rEV598, Revlimid, Rhenium-186-Etidronat, Rituximab, Roferon-A, Romurtid, Roscovitin, Rubitecan, Sagopilone, Salagen, Sandostatin, Sargramostim, Satraplatin, Seocalcitol, Semustin, Sirolimus, Sizofiran, Sizofilan, Sobuzoxan, Solu-Medrol, BAY 43-9006 (Sorafenib), Squalamin, Streptozocin, Strontium-89-chlorid, Sunitinib, Synthroid, T-138067, Tabi, Tafluposid, Tamoxifen, Tamoxifencitrat, Tamsulosin, Tarceva, Tasonermin, Tastolacton, Tastuzumab, Taxoter, Taxoprexin, Tazaroten, Teceleukin, Tegafur, Telatinib, Temsirolimus, Temozolomid, Teniposid, Teosulfan, Testolacton, Testosteron-Propionat, Testred, Thalidomide, TheraCys, Thioguanin, Thiotepa, Thymalasin, Thymosin-alpha-1 , Thyrotropin, Tiazofurin, Ticilimumab, Tiludronsaure, Tipifarnib, Tirapazamin, Tiuxetan, TLK-286, Topotecan, Toremifen, Tositumomab, Topotecan, TransMID-107R, Trastuzumab, Tratuzumab, Tretinoin,

Trelstar, Tretinoin, Trexall, Triapine, Trimethylmelamin, Trimetrexat, Triptorelin-Acetat, Triptorelin-Pamoat, Ubenimex, UFT, Ukrain, Uridin, Valrubicin, Valspodar, Vapreotid, Vatalanib, Vectibix, Velcade, Verteporfin, Vesnarinon, Vidarabin, Vimlizin, Vinblastin, Vincristin, Vindesin, Vinflunin, Vinorelbin, Vimlizin, Vitaxin, Volociximab, Vorinostat, Xaliprode, Z-100, Zactima, Zindol, Zinecard, Zinostatin-Stimalamer, Zofran and Zoledron acid.

Of selective interest are those combinations of antibodies that can be used for the prevention and treatment of antiestrogen-resistant breast cancer together with aromatase inhibitors, antiestrogens, tamoxifen and other SERM'S, GnRH analogs and antagonists, EGFR inhibitors, Her-2/neu inhibitors, MAPK inhibitors and IGFIR inhibitors.

Suitable antiestrogens which can be combined together with the instant PRLR antibodies, are the following compounds:

1 1 -Fluoro-7a-{5-[methyl(7,7,8,8,9,9,9-heptafluorononyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol N-oxide

1 1 -Fluoro-7a-{5-[methyl(8,8,9,9, 10, 10, 10-heptafluorodecyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol N-oxide

(RS)-1 1 -Fluoro-7a-{5-[methyl(7, 7, 8,8,9,9, 10, 10, 10-nonafluorodecyl)amino]-pentyl}- 17a-methylestra-1 ,3,5(10)-triene-3, 17 -diol N-oxide

1 1 -Fluoro-7a-{5-[methyl(8,8,9,9,9-pentafluorononyl)amino]pentyl}-17a-methylestra- 1 ,3,5(10)-triene-3, 17 -diol N-oxide 11 -Fluoro-7a-{5-[methyl(9,9, 10,10,10-pentafluorodecyl)amino]pentyl-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol N-oxide

11 -Fluoro-7a-{5-[methyl(8,8,9,9,9-pentafluorononyl)amino]pentyl}-17a-methylestra- 1, 3,5(10)-triene-3,17 -diol

11 -Fluoro-7a-{5-[methyl(7, 7, 8,8,9,9, 10,10,10-nonafluorodecyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(7,7,8,8,9,9,9-heptafluorononyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol

17a-Ethinyl-11 -fluoro-7a-{5-[methyl(7,7, 8,8,9,9, 10,10,1 O-nonafluorodecyl)- amino]pentyl}-estra-1 ,3,5(10)-triene-3, 17β-άϊοΙ

17a-Ethinyl-11 -fluoro-3-(2-tetrahydropyranoyloxy)-7a-{5-

[methyl(7,7, 8,8,9,9, 10, 10, 10-nonafluorodecyl)amino]pentyl}-estra-1 ,3,5(10)-trien-17β ol

11 -Fluoro-3-(2-tetrahydropyranyloxy)-7a-{5-[methyl(7, 7, 8,8,9,9, 10,10,10- nonafluorodecyl)amino]pentyl}-17a-methylestra-1 ,3,5(10)-trien-17β-οΙ

11 -Fluoro-7a-{5-[methyl(7, 7, 8,8,9,9, 10, 10, 10-nonafluorodecyl)amino]pentyl}-17a- trifluoromethylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(6,6,7,7,8,8,8-heptafluorooctyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(8,8,9,9, 10,10,10-heptafluorodecyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(6,6,7, 7, 8,8,9,9, 10,10,10-undecafluorodecyl)amino]-pentyl}- 17a-methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(5,5,6,6,7,7,8,8,8-nonafluorooctyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(9,9, 10,10,11,11,11 -heptafluoroundecyl)amino]-pentyl}-17a methylestra-1 ,3,5(10)-triene-3, 17 -diol

11 -Fluoro-7a-{5-[methyl(9,9, 10,10,10-pentafluorodecyl)amino]pentyl}-17a- methylestra-1 ,3,5(10)-triene-3, 17 -diol.

These compounds show high antiestrogenic activity after administration. Due to the blockade of the 17B-position formation of (active) metabolites is suppressed. The especially preferred antiestrogenic compounds that can be combined are 1 1 β- Fluoro-17a-methyl-7a-{5-[methyl(8, 8,9,9, 9-pentafluorononyl)amino]pentyl}-estra- 1 , 3,5(10)-triene-3, 17 -diol and

7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]-estra-1 ,3,5(10)-triene-3, 17 beta- diol, and their pharmaceutically acceptable derivatives or analogues thereof.

Further interesting steroidal antiestrogenic compounds which can be combined with the instant PRLR antibodies are, for example, tamoxifen, raloxifene, droloxifen, toremifen, lasofoxifen, arzoxifen, GW5638 *), EM-800 **), idoxifen and basedoxifene TAS-108 (Yamamoto et al. , Clin. Cancer Res. 315, Vol. 1 1 , 315-322, 2005), which has the following structure

and ICI 164.384 (Wakeling et al. , Cancer Res. 51 , 3867, (1991 ).

Suitable aromatase inhibitors which can be combined together with the instant PRLR antibodies are for example aminoglutethimide, fadrozole, anastrozole, letrozole, vorozole, formestane, exemestane and atamestane

Preferably, the instant PRLR antibodies can be used either alone as monotherapy or in combination with one or more of tamoxifen; faslodex; the aromatase inhibitors aminoglutethimide, fadrozole, anastrozole, letrozole, vorozole, formestane,

exemestane and atamestane; GnRH analogues or antagonists, such as buserelin, goserelin, leuprorelin, nafarelin, triptorelin, and cetrorelix, EGFR inhibitors, such as erlotinib, cetuximab, gefitinib, and panitumumab ; Her-2/neu inhibitors, such as trastuzumab and pertuzumab; IGFI inhibitors and MAPK inhibitors, such as U0126 or in a sequential manner with the mentioned medicaments. Where separate dosage formulations are used, the instant antibody and one or more additional therapeutic agents may be administered at essentially the same time or sequentially.

Commonly known and used adjuvants, as well as further suitable carriers or diluents may be used.

Suitable carriers and adjuvants may be such as recommended for pharmacy, cosmetics and related fields in: Ullmann's Encyclopedia of Technical Chemistry, Vol. 4, (1953), pp. 1 -39; Journal of Pharmaceutical Sciences, Vol. 52 (1963), p. 918ff;

H.v.Czetsch-Lindenwald, "Hilfsstoffe fur Pharmazie und angrenzende Gebiete";

Pharm. Ind. 2, 1961 , p.72ff; Dr. H. P. Fiedler, Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, Cantor KG, Aulendorf in Wurttemberg, 1971 .

The instant antibodies can be applied in an oral, parenteral, e.g. intraperitoneal, intravenous, intramuscular, subcutaneous or percutaneous form. The instant antibodies can also be implanted into tissue.

The instant antibodies can also be applied as pharmaceutical compositions, which can be prepared by known methods of preparing galenics for oral, parenteral, e.g.

intravenous, intraperitoneal, intramuscular, subcutaneous or percutaneous application.

The antibodies can be administered in the form of tablets, pills, dragees, gel capsules, granules, suppositories, implants, injectable sterile aqueous or oily solutions, suspensions or emulsions, ointments, creams, gels, patches for transdermal administration, or formulations suitable for administration by inhalation, for instance nasal sprays.

The amounts (a "pharmaceutically effective amount") of the combined active agents to be administered vary within a broad range and depend on the condition to be treated and the mode of administration. They can cover any amount efficient for the intended treatment. Determining a "pharmaceutically effective amount" of the combined active agent is within the purview of a person skilled in the art. The weight ratio of the instant antibodies when used for the treatment and prevention of antiestrogen-resistant breast cancer can vary within a broad dose range. An acceptable range is between 0.1 to 300 mg, preferably between 0.1 to 200 mg, more preferably between 0.1 to 150 mg, much more preferably between 0.1 to 100 mg and most preferably between 10 and 150 mg.

In a combined use of the instant antibodies together with cytotoxic agents they can either be present in equal amounts or one component can be present in excess of the other components. Preferably, 0.1 to 300 mg of the antibodies or/ and 0.1 to 100 mg of the cytotoxic agent, both are administered in a unit dose, more preferably in a unit dose of 0.1 to 200 mg of each of the antibodies or/ and cytotoxic agent, acceptable more preferably in a unit dose of 0.1 to 150 mg of each of the antibodies or/ and cytotoxic agent, much more preferably in a unit dose of 0.1 to 100 mg of each of the antibodies or/ and cytotoxic agent, and most preferably in a unit dose of 10 to 150 mg of each of the antibodies or/ and cytotoxic agent.

In special cases up to 300 mg of the instant antibody may be administered.

The instant antibodies and the cytotoxic agents can be administered either together or separately, at the same time and/sequentially. Preferably they are administered combined in one unit dose. In case they are administered sequentially, preferably the antibody is administered before the cytotoxic agent, as defined above.

DESCRIPTION OF THE FIGURES

Figure 1 shows the effects of specific neutralising neutralizing PRLR antibodies that are effective in the preventive setting of a model of antiestrogen resistant breast cancer

Figure 2 shows the effects of a specific neutralizing PRLR antibody that is effective in the therapeutic setting of a model of antiestrogen resistant breast cancer

The invention is further illustrated in the Examples. The following Examples are, however, not to be understood as a limitation.

EXAMPLES

Example 1

Neutralizing PRLR antibodies are effective in a mouse model of antiestrogen-resistant breast cancer (preventive setting)

8 week old female SHN mice received a pituitary isograft under the kidney capsule (Fig. 1 B-F) or remained unoperated (Fig. 1 A). Group size was 10 animals per group. Pituitary isografted animals remained either untreated (Fig. 1 B) or were treated with unspecific control antibody (once weekly intraperitoneal, Fig. 1 E), or with specific antibody lgG2a 005_C04 (30 mg/kg, once weekly intraperitoneal, Fig. 1 F) or received subcutaneous injections of either ICI (faslodex, 5 mg/kg dissolved ethanol/arachisoil (10%/90%), 6 days per week, Fig. 1 C) or cetrorelix (100 g, dissolved in 5% mannitol in water, 6 days per week, Fig. 1 D). Treatment with antibodies and compounds was performed from day 1 1 -72 after pituitary isografting, autopsy was performed on day 73.

Mammary gland whole mounts were prepared after fixation in Carnoy's solution and staining with carmine alaun and are shown in Figure 1 .

Hyperplastic alveolar nodules (HANs) that resembles preneoplastic mammary gland growth that will give rise to breast cancer in mice are indicated by black arrows in Figure 1 . HANs are observed after pituitary isografting, however detection is a bit difficult since the enhanced alveolar outgrowth of the normal mammary gland tissue hides them (Fig. 1 B). Treatment wth ICI or cetrorelix inhibits alveolar differentiation of normal mammary gland tissue, whereas the growth of preneoplastic HANs remains unaffected. HANs are therefore easier to detect after ICI and cetrorelix treatment (Fig. 1 C, D) since both agents inhibit normal mammary gland growth (i.e. sidebranching and alveolar development). In the SHN breast tumor model, HANs represent an example of antiestrogen-resistant breast tumors since ICI and cetrorelix cannot prevent them. Specific PRLR antibodies (i.e. lgG2a 005_C04, Fig. 1 F) but not unspecific antibodies (Fig. 1 E) inhibit the growth of preneoplastic HANs.

Neutralising PRLR antibodies are therefore suitable to prevent antiestrogen-resistant breast cancer.

Example 2

Neutralizing PRLR antibodies are effective in a mouse model of antiestrogen- resistant breast cancer (therapeutic setting)

8 week old female SHN mice received a pituitary isograft under the kidney capsule or remained unoperated. Group size was n=8-1 1 animals per group. Treatment of the animals started either on day 14 or day 71 after pituitary isografting and was performed till day 127 after pituitary isografting. Animals were sacrificed on day 128 after pituitary isografting and mammary gland whole mounts were prepared as described in Example 1 .

Example 2 encompasses the following experimental groups:

1 ) unoperated animals killed on day 128 (Fig. 2A), n=1 1

2) isografted animals killed on day 128 after pituitary isografting (Fig. 2B), n=1 1

3) isografted animals treated with faslodex (5 mg/kg subcutaneously, 6 times per week) from day 71 -127 after pituitary isografting and killed on day 128 (Fig. 2C), n=1 1

4) isografted animals treated with specific PRLR antibody (lgG2a 005_C04, 30 mg/kg, subcutaneously, once weekly) from day 71 -127 after pituitary isografting and killed on day 128 (Fig. 2D), n=1 1

5) isografted animals treated with faslodex (5 mg/kg subcutaneously, 6 times weekly) from day 14-70 after pituitary isografting and then treated with specific PRLR antibody (lgG2a 005_C04, 30 mg/kg, once weekly, subcutaneously) from day 71 -127 and killed on day 128 (Fig. 2E), n=10

6) isografted animals treated with specific PRLR antibody (lgG2a 005_C04, 30 mg/kg, once weekly, subcutaneously) from day 14-70 after pituitary isografting and then treated with faslodex (5 mg/kg subcutaneously, 6 times weekly) from day 71 -127 and killed on day 128 (Fig. 2F), n=10

7) isografted animals treated with faslodex (5 mg/kg, subcutaneously, 6 times

weekly) from day 14-127 after pituitary isografting and killed on day 128 (Fig. 2G), n=9

8) isografted animals treated with specific PRLR antibody (lgG2a 005_C04, 30 mg/kg, subcutaneously, once weekly) from day 14-127 after pituitary isografting and killed on day 128 (Fig. 2H), n=10

Hyperplastic alveolar nodules (HANs) that resemble preneoplastic mammary gland growth that will give rise to breast cancer in mice are indicated by black arrows in

Figure 2. When autopsy was performed on day 128 instead of day 73 (Figure 1 ), more untreated and unoperated animals (8 out of 1 1 animals) developed small HANs (Figure 2A). In pituitary-isografted untreated animals there were more and larger HANs (Figure 2B) compared to untreated unoperated animals (Figure 2A), all pituitary- grafted untreated animals (1 1 out of 1 1 ) showed HANs. In Example 1 it was

demonstrated that treatment with the antiestrogen faslodex could not prevent HANs (treatment was performed from dayl 1 -72 after pituitary grafting) and HANs were present in faslodex-treated animals on day 73 after pituitary grafting. In this

experiment, treatment was performed in the therapeutic setting, e.g. treatment started on day 71 (when HANs were already developed in pituitary isografted animals as known from Example 1 , Figure 1 ). Faslodex was ineffective in the therapeutic setting (Figure 2C), all faslodex-treated animals (1 1 out of 1 1 ) were positive for HANs. In sharp contrast HANs were not visible in the majority of animals when treated in the therapeutic setting with lgG2a 005_C04 (Figure 2D). Only 2 out of 1 1 animals showed extremely small and very few HANs after treatment with neutralising PRLR antibody, thus demonstrating efficacy of lgG2a 005_C04 in the therapeutic setting of the experiment. When animals were treated from day 14-70 with faslodex and from day 71 -127 with neutralising PRLR antibody, only 1 out of 10 animals showed few and small HANs, whereas the all other animals were devoid of HANs (Figure 2E). This demonstrates the therapeutic efficacy of PRLR antibodies in antiestrogen-resistant breast cancer, because from Example 1 (Figurel ) it is known that after faslodex treatment from day 1 1 -72 animals develop multiple and large HANs. Neutralising PRLR antibodies are capable of treating such HANs when treatment starts on day 71 and is performed till day 127. In contrast, when antibody treatment was performed from day 14-70 and followed by faslodex treatment from day 71 -127 five out of 10 animals showed small HANs (Figure 2F). This demonstrates that although PRLR antibodies prevent HANs when given form day 1 1 -72 (Example 1 , Figure 1 ), subsequent faslodex treatment is ineffective and gives rise to HANs again (Figure 2F). Faslodex treatment on day 14-127 leads to HANs in all animals (9 out of 9, Figure 2G) again demonstrating that faslodex in a preventive setting is without any effect. PRLR antibody lgG2a 005_C04 given on day 14-127 leads to only one single HAN in one animal out of 1 1 , whereas all other PRLR antibody treated animals are devoid of HANs (Figure 2H) again showing the efficacy of a PRLR antibody in a preventive setting within this model.

In conclusion the experiment shown in Figure 2 and described in this example demonstrates efficacy of PRLR antibodies with regard to HAN prevention (Figure 2H) and treatment (Figure 2D, E). Faslodex as antiestrogen shows no efficacy with regard to HAN prevention (Figure 2G) and treatment (Figure 2C, F).

Neutralising PRLR antibodies are therefore suitable to treat antiestrogen-resistant breast cancer.

Example 3

Comparison of known antibodies with instant antibodies

To analyze the in vitro efficacy of the neutralizing instant PRLR antibodies compared to the known antibody HE06.642 as of WO 2008/022295, the inhibition of prolactin- activated cellular proliferation of BaF3 cells was used. The cells were stably

transfected with human PRLR and were routinely cultured in RPMI containing 2 mM glutamine in the presence of 10% FCS and 10 ng/ml of human prolactin. After six hours of starvation in prolactin-free medium containing 1 % FCS, cells were seeded into 96-well plates at a density of 10000 cells per well. Cells were stimulated with 20 ng/ml prolactin and coincubated with increasing doses of neutralizing instant PRLR antibodies or the known antibody HE06.642 for two days. Cellular proliferation was analyzed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega). Dose- response curves for the inhibition of prolactin-stimulated cellular growth were generated and IC50 values calculated. As negative control, stimulation with an unspecific control antibody was used. The IC50 values are depicted in the following Table 3.

Table 3

As it can be seen from the results, with the exception of antibody 005-C04, the instant PRLR antibodies are more potent inhibitors of the human PRLR than the known antibodies.

This is an advantage of the instant PRLR antibodies since lower doses compared to the known antibody HE06.642 will be required. Lower doses reduce the immunogenic risk of the antibodies for the patient.

Claims

What is claimed is:

1 . Use of neutralizing prolactin receptor antibodies as medicament for the treatment of antiestrogen-resistant breast cancer.

2. Use of neutralizing prolactin receptor antibodies as medicament for the prevention of antiestrogen-resistant breast cancer.

3. Use according to claim 1 or 2, characterized in that the antibodies comprise sequence Seq. ID No.1 to sequence Seq. ID No. 518.

4. Use according to any one of claiml to 3, characterized in that the antibodies are combined with one or more cytotoxic agents.

5. Use according to any of claiml to 4, characterized in that the antibodies are combined with one or more of aromatase inhibitors, antiestrogens, tamoxifen and other SERM'S, GnRH analogs and antagonists, EGFR inhibitors, Her-2/neu inhibitors, MAPK inhibitors and IGFIR inhibitors.

6. Use according to any of claimsl to 5 together with suitable diluents and/ or carriers.

7. Use according to any of claims 1 to 6, characterized in that the antibody is applied in a range between 0.1 to 300 mg.

8. Use according to any of claims 1 to 6, characterized in that the antibody is applied in a range between 0.1 to 200 mg.

9. Use according to any of claims 1 to 6, characterized in that the antibody is applied in a range between 0.1 to 150 mg.

10. Use according to any of claims 1 to 6, characterized in that the antibody is applied in a range between 0.1 and 100 mg. Use according to any of claims 1 to 6, characterized in that the antibody ^' applied in a range between 10 to 150 mg.