Neutralizing prolactin receptor antibodies and their therapeutic use

Abstract

The present invention is directed to the neutralizing prolactin receptor antibody 006-H08, as well as maturated forms thereof, and antigen-binding fragments, pharmaceutical compositions containing them and their use in the treatment or prevention of benign disorders and indications mediated by the prolactin receptor such as endometriosis, adenomyosis, non-hormonal female contraception, benign breast disease and mastalgia, lactation inhibition, benign prostate hyperplasia, fibroids, hyper- and normoprolactinemic hair loss, and cotreatment in combined hormone therapy to inhibit mammary epithelial cell proliferation. The antibodies of the invention block prolactin receptor-mediated signaling.

Claims

1. Antibody 006-H08, 006-H08-12-2, 006-H08-13-2, 006-H08-13-6-1, 006-H08-14-6-0, 006-H08-15-5, 006-H08-19-1, 006-H08-29-1, 006-H08-32-2, 006-H08-33-0, 006-H08-33-16-0, 006-H08-35-17-1, 006-H08-35-17-4, 006-H08-35-1, 006-H08-36-17-0, 006-H08-37-19-0, 006-H08-39-7, 006-H08-48-5, 006-H08-53-27-0, 006-H08-59-30-0, 006-H08-63-32-4, 006-H08-65-33-2, or 006-H08-68-35-2 or an antigen-binding fragment thereof, which antigen-binding fragment antagonizes prolactin receptor-mediated signaling and which binds to an epitope of the extracellular domain (ECD) of the prolactin receptor PRLR or to the ECD of polymorphic variant I146L of PRLR or to the ECD of polymorphic variant I76V of PRLR, the amino acid sequence of the ECD of PRLR corresponding to SEQ ID NO: 70.

2. An antibody, or antigen-binding fragment thereof that antagonizes prolactin receptor-mediated signaling and that binds to an epitope of the extracellular domain (ECD) of the prolactin receptor PRLR or to the ECD of polymorphic variant I146L of PRLR or to the ECD of polymorphic variant I76V of PRLR, whereby the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7 and 13 and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, and 29, or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 78, 24, 90; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 75, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 82, 24, 91; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 82, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 86, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 87, 24, 100; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 87, 24, 92; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 89, 24, 93; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 79, 24, 101; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 76, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 89, 24, 90; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 100; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 97; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 98; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 83, 24, 99; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 96; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 94; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 88, 24, 90; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 74, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 81, 24, 95; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 75, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 77, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 18, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 80, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 85, 24, 29; or the variable heavy chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 1, 7, 13, and the variable light chain of the antibody or antigen-binding fragment thereof comprises the CDR sequences corresponding to SEQ ID NO: 84, 24, 29.

3. Antibody, or antigen-binding fragment according to claim 2, whereby the antibody, or antigen-binding fragment thereof comprises a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 34, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 40, or a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 143, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 165, or a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 144, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 166, or a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 145, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 167, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 146, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 168, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 147, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 169, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 148, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 170, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 149, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 171, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 150, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 172, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 151, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 173, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 152, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 174, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 153, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 175, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 154, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 176, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 155, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 177, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 156, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 178, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 157, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 179, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 158, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 180, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 159, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 181, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 160, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 182, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 161, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 183, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 162, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 184, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 163, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 185, a variable heavy chain domain with an amino acid sequence according to SEQ ID NO: 164, and a variable light chain domain with an amino acid sequence according to SEQ ID NO: 186.

4. Antibody, or antigen-binding fragment thereof according to claim 2, whereby the antibody, or antigen-binding fragment thereof is immunospecific for an epitope between amino acid position 1 to 210 of PRLR.

5. Antibody, or antigen-binding fragment thereof according to claim 2 wherein the heavy constant chain thereof is a heavy constant chain from IgG1, IgG2, IgG3 or IgG4.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Expression of prolactin-mRNA (PRL-mRNA) (analyzed by real-time TaqMan PCR analysis) in human endometrium and lesions (ectopic tissue) from healthy women and women suffering from endometriosis.

(2) FIG. 2: Expression of prolactin receptor-mRNA (PRLR-mRNA) (analyzed by real-time TaqMan PCR analysis) in human endometrium and lesions (ectopic tissue) from healthy women and women suffering from endometriosis.

(3) FIG. 3: Northern blot analysis of PRLR gene expression in rat tissues. Gene expression of the PRLR revealed high expression in placenta and prostate.

(4) FIG. 4: Western blot analysis of PRLR expression in rat prostates treated with different hormones. Estradiol treatment of intact rats and castration lead to an upregulation of PRLR protein in rat prostates whereas dihydrotestosterone treatment of intact rats had no impact on PRLR expression in the prostate compared to vehicle treatment of intact animals.

(5) FIG. 5: Inhibition of prolactin-activated Ba/F (=Baf) cell proliferation (stably expressing the human PRLR) by neutralizing PRLRantibodies and unspecific control antibodies. The IC.sub.50 values were determined for the following antibodies in IgG1 format: 005-C04 (closed circles): 1.29 g/ml=8.6 nM; 006-H08 (open circles): 0.15 g/ml=1 nM; HE06.642 (closed triangles): 0.34 g/ml=2.2 nM; 002-H06 (open triangles): 0.54 g/ml=3.6 nM; 002-H08 (closed squares): 0.72 g/ml=4.8 nM; unspecific control antibody (open squares): no inhibition of cell proliferation

(6) FIG. 6: Inhibition of prolactin-induced rat lymphoma cell proliferation (NB2 cells) by neutralizing PRLR antibodies and unspecific control antibodies. The following IC.sub.50 values were determined: XHA06.642 (closed circles): 10 g/ml=67 nM; XHA06.983 (open circles): no effect on rat lymphoma cell proliferation; unspecific control antibody (closed triangle): no effect at 10 g/ml.

(7) FIG. 7: Inhibition of prolactin-stimulated STAT5 phosphorylation in T47D cells by neutralizing PRLR antibodies and unspecific control antibody.

(8) The unspecific control antibody (FITC) does not inhibit STAT5 phosphorylation in T47D cells. In contrast the antibodies XHA06.642, 005-C04 (=IgG1 005-C04), and 006-H08 (=IgG1 006-H08) inhibit in a dose-dependent manner phosphorylation of STAT5 in T47D cells.

(9) FIG. 8: Effects of neutralizing PRLR antibodies and unspecific controls on prolactin-activated luciferase reporter gene activity using HEK293 cells stably transfected with the human prolactin receptor (hPRLR) and transiently expressing the luciferase gene under the control of lactogenic hormone response elements (LHREs). The IC.sub.50 values were determined for the following antibodies in IgG1 format: 006-H08 (closed circles): 0.83 g/ml=5.5 nM; HE06.642 (open circles): 0.63 g/ml=4.2 nM; unspecific control antibody (closed triangle): no inhibition of luciferase activity.

(10) FIG. 9: Effects of neutralizing PRLR antibodies and unspecific controls on prolactin-activated luciferase reporter gene activity using HEK293 cells stably transfected with the murine prolactin receptor (mPRLR) and transiently expressing the luciferase gene under the control of lactogenic hormone response elements (LHREs). The IC.sub.50 values were determined for the following antibodies in IgG1 format: 005-C04 (closed triangles): 0.45 g/ml=3 nM; XHA06.642 (closed circles): >>50 g/ml>>333 nM, unspecific control antibody (open circles): no inhibition of luciferase activity.

(11) FIG. 10: Inhibition of prolactin-activated Ba/F (=Baf) cell proliferation (stably expressing the murine prolactin receptor) by neutralizing prolactin receptor antibodies and unspecific control antibodies. The IC.sub.50 values were determined for the following antibodies in IgG1 format: unspecific FITC antibody (closed squares): no inhibition of cell proliferation; HE06.642 (closed circles): >>>30 g/ml>>>200 nM; 001-E06 (open circles): 43.7 g/ml=291 nM; 001-D07 (closed triangles): 16.5 g/ml=110 nM; 005-C04 (open triangles): 0.74 g/ml=4.9 nM.

(12) FIG. 11: Pregnancy rates and mean litter size in female mice treated with phosphate-buffered saline (=vehicle), unspecific control antibody (FITC IgG1) or neutralizing antibody IgG1 005-C04 (=005-C04). Pregnancy rates were 87.5% (vehicle treated females), 75% (females treated with 10 mg/kg unspecific antibody), 100% (females treated with 10 mg/kg IgG1 005-C04), and 0% (females treated with 30 mg/kg IgG1 005-C04). Mean litter size was 10.9 animals (vehicle treated females), 12.3 animals (females treated with 10 mg/kg unspecific antibody), 13 animals (females treated with 10 mg/kg IgG1 005-C04) and 0 animals (females treated with 30 mg/kg IgG1 005-C04).

(13) FIGS. 12A and 12B: Kabat Numbering of framework amino acid positions according to Johnson and Wu (Nucleic Acids Res. 2000, 28, 214-218).

(14) FIG. 13: FACS analysis results with selected anti-PRLR antibodies (005-C04, 001-E06, HE06642). Binding of the antibodies was determined at a fixed concentration on HEK293 cells expressing the human and mouse PRLR in comparison to the parental cell line not expressing PRLR.

(15) FIG. 14A: Litter weight gain for each postpartal day expressed as percentage of litter weight obtained on postpartal day 1. Weight gain of litters from untreated mothers (closed circles), from mothers treated with 10 mg/kg unspecific murine IgG2a antibody (open circles), and from mothers treated with the neutralizing antibody 005-C04 containing murine IgG2a constant domains (=IgG2a 005-C04) at 10 mg/kg (closed triangles) and at 30 mg/kg (open triangles) is shown. Arrows indicate days on which antibody injection was performed. There is a significant reduction in weight gain from litters of mothers treated with 30 mg/kg IgG2a 005-C04 from postpartal day 8 onwards.

(16) FIG. 14B: Incremental litter weight gain from day to day expressed as percentage of litter weight on postpartal day 1. Results from litters of untreated mothers (closed circles), mothers treated with 10 mg/kg unspecific murine IgG2a antibody (open circles), mothers treated with the neutralizing antibody 005-C04 containing murine IgG2a constant domains (=IgG2a 005-C04) at 10 mg/kg (closed triangles) and at 30 mg/kg (open triangles) are shown. Basically FIG. 14A presents the slope of the graphs shown in FIG. 14A. Daily weight gain in litters from untreated mothers and mothers treated with 10 mg/kg unspecific antibody oscillates around 30% of the litter weight on postpartal day 1. In contrast, treatment of mothers with 30 mg/kg IgG2a 005-C04 leads to a significant reduction in weight gain from day 7 onwards (*p<0.05;***p<0.005 vs. litters from mothers treated with unspecific antibody) whereas treatment with 10 mg/kg IgG2a 005-C04 leads to a significant reduction in daily weight gain from day 11 onwards (p<0.05 vs. litters from mothers treated with unspecific antibody). Arrows indicate days of antibody application.

(17) FIG. 14C: Histological sections from mammary glands of lactating mothers. Mammary glands from untreated mothers or mothers treated with unspecific antibody are filled with ducts producing milk. In contrast mammary gland involution, evidenced by the appearance of fatty islands (black arrows), is induced dose-dependently by the neutralizing IgG2a 005-C04 antibody.

(18) FIG. 14D: Milk protein expression in mammary glands from lactating mothers. Expression of the milk proteins beta casein (Csn-2), whey acidic protein (WAP), and IGF-1 is reduced in a dose-dependent manner in mothers treated with neutralizing PRLR antibody IgG2a 005-C04, but not with unspecific antibodies. Gene expression was normalized to the expression of TATA box binding protein (TBP).

(19) FIG. 15A: Formation of side branches and alveolar like structures in a hyperprolactinemic mouse model of benign breast disease. The neutralizing PRLR antibody IgG1 005-C04 (=005-C04) inhibits side branching and the formation of alveolar like structures at 10 and 30 mg/kg in mice that received a pituitary isograft.

(20) FIG. 15B: Extent of epithelial hyperplasia and epithelial cell proliferation in a hyperprolactinemic mouse model of benign breast disease. Some BrdU-positive cells are marked by white arrows. The neutralizing PRLR antibody IgG1 005-C04 (=005-C04) blocks epithelial hyperplasia and epithelial cell proliferation in the mammary gland.

(21) FIG. 15C: Extent of STAT5 phosphorylation in a hyperprolactinemic mouse model of benign breast disease. Some phospho-STAT5-positive cells are indicated by white arrows. The neutralizing PRLR antibody IgG1 005-C04 (=005-C04) completely blocks STAT5 phosphorylation when applied at a dosage of 30 mg/kg.

(22) FIG. 16: Inhibition of prostate growth by the neutralizing PRLR antibody 005-C04 containing murine IgG2a constant domains (=IgG2a 005-C04). Pituitary isografting stimulates prostate growth in comparison to untreated sham-operated mice. Treatment with neutralizing PRLR antibodies at doses of 10 mg/kg and at doses of 30 mg/kg inhibits prostate growth (***p<0.005 vs. untreated, sham-operated mice).

(23) FIG. 17: Neutralizing PRLR antibodies stimulate hair growth in the presence of hyperprolactinemia. Photographs were taken three weeks after pituitary isografting (and shaving) from male mice used in the experiments described in Example 17 and in FIG. 16. Hyperprolactinemia inhibits hair regrowth in the shaved areas. Neutralizing PRLR antibodies, but not unspecific antibodies stimulate hair regrowth under hyperprolactinemic conditions at doses of 10 and 30 mg/kg of 005-C04 (=IgG2a 005-C04).

(24) FIG. 18: Neutralizing PRLR antibodies but not unspecific antibodies stimulate hair regrowth in shaved areas in hyper- and normoprolactinemic male and female mice (Example 18). Neutralizing PRLR antibodies are therefore suitable for the treatment of hair loss under normo- and hyperprolactinemic conditions in men (FIG. 18 B) and women (FIG. 18A).

(25) FIG. 19: Neutralizing PRLR antibodies but not unspecific control antibodies inhibit enhanced epithelial cell proliferation in the mammary gland after combined hormone therapy, i.e. combined estrogen plus progestin therapy.

(26) The absolute number of proliferating ductal epithelial cells within 4 cross-sections of the mammary gland was evaluated and the medians are depicted as horizontal bars within the figure. Epithelial cell proliferation in ovariectomized, vehicle treated mice is rather low (median=0). Estradiol treatment leads to some stimulation of epithelial cell proliferation (median=9), maximal mammary epithelial cell proliferation is observed under estrogen plus progesterone treatment (median=144). Treatment with neutralising prolactin receptor antibody 005-C04 (median=84 after treatment with 10 mg/kg 005-C04; median=27 after treatment with 30 mg/kg 005-C04) but not with unspecific control antibody (median=154) leads to a dose-dependent decrease in mammary epithelial cell proliferation almost back to estradiol-only levels.

(27) Neutralising PRLR antibodies are therefore suitable to treat enhanced mammary epithelial cell proliferation under combined hormone therapy, i.e. estradiol plus progesterone treatment.

(28) FIG. 20: Neutralizing PRLR antibodies but not unspecific control antibodies inhibit endometriosis interna in mice. The results are depicted as disease scores as described in Example 20. The median disease score for each experimental group is indicated as a horizontal bar. Normoprolactinemic mice develop endometriosis interna to some degree (median disease score=0.25). Hyperprolactinemia due to pituitary isografting enhances the disease score and more animals suffer from the disease (median disease score=2.5). Whereas treatment with 30 mg/kg unspecific antibody once (median score=2.5) or twice (median score=2) weekly had no influence on the disease, treatment with specific neutralizing antibodies shows a dose-dependent decrease in the amount of sick animals; the median disease score in all cases in which specific antibody was used was zero. Notably, all animals receiving either 10 or 30 mg/kg specific antibody twice weekly were completely cured and their disease score was significantly lower than the disease score of normoprolactinemic mice. Neutralising PRLR antibodies are therefore suitable to treat endometriosis interna (=adenomyosis uteri) and endometriosis externa in women.

(29) FIGS. 21A and 21B: Binding tests of maturated 006-H08 Fab variants using homogenous time-resolved fluorescence (HTRF) assay:

(30) Fab-containing E. coli supernatants were tested for binding to the extracellular domain of the human PRLR via competition to the IgG1 molecules of 006-H08. The figure illustrates the binding of the Fab variants as a bar diagram. The signal intensities (at 665 nM) at five different incubation times are given on the y-axes, the names of the Fab variants on the x-axes. Lower signals compared to the control Fab 006-H08 at a given time point indicate improved binding to PRLR. All Fabs listed in Part 1 represent improved binders, while in Part 2 improved as well as some non-improved binders are shown.

(31) Seq ID NO:1 represents amino acid sequence of HCDR1, 006-H08

(32) Seq ID NO:2 represents amino acid sequence of HCDR1, 002-H06

(33) Seq ID NO:3 represents amino acid sequence of HCDR1, 002-H08

(34) Seq ID NO:4 represents amino acid sequence of HCDR1, 006-H07

(35) Seq ID NO:5 represents amino acid sequence of HCDR1, 001-E06

(36) Seq ID NO:6 represents amino acid sequence of HCDR1, 005-C04

(37) Seq ID NO:7 represents amino acid sequence of HCDR2, 006-H08

(38) Seq ID NO:8 represents amino acid sequence of HCDR2, 002-H06

(39) Seq ID NO:9 represents amino acid sequence of HCDR2, 002-H08

(40) Seq ID NO:10 represents amino acid sequence of HCDR2, 006-H07

(41) Seq ID NO:11 represents amino acid sequence of HCDR2, 001-E06

(42) Seq ID NO:12 represents amino acid sequence of HCDR2, 005-C04

(43) Seq ID NO:13 represents amino acid sequence of HCDR3, 006-H08, 002-H06

(44) Seq ID NO:14 represents amino acid sequence of HCDR3, 002-H08

(45) Seq ID NO:15 represents amino acid sequence of HCDR3, 006-H07

(46) Seq ID NO:16 represents amino acid sequence of HCDR3, 001-E06

(47) Seq ID NO:17 represents amino acid sequence of HCDR3, 005-C04

(48) Seq ID NO:18 represents amino acid sequence of LCDR1, 006-H08

(49) Seq ID NO:19 represents amino acid sequence of LCDR1, 002-H06

(50) Seq ID NO:20 represents amino acid sequence of LCDR1, 002-H08

(51) Seq ID NO:21 represents amino acid sequence of LCDR1, 006-H07

(52) Seq ID NO:22 represents amino acid sequence of LCDR1, 001-E06

(53) Seq ID NO:23 represents amino acid sequence of LCDR1, 005-C04

(54) Seq ID NO:24 represents amino acid sequence of LCDR2, 006-H08, 002-H08

(55) Seq ID NO:25 represents amino acid sequence of LCDR2, 002-H06

(56) Seq ID NO:26 represents amino acid sequence of LCDR2, 006-H07

(57) Seq ID NO:27 represents amino acid sequence of LCDR2, 001-E06

(58) Seq ID NO:28 represents amino acid sequence of LCDR2, 005-C04

(59) Seq ID NO:29 represents amino acid sequence of LCDR3, 006-H08

(60) Seq ID NO:30 represents amino acid sequence of LCDR3, 002-H06, 001-E06

(61) Seq ID NO:31 represents amino acid sequence of LCDR3, 002-H08

(62) Seq ID NO:32 represents amino acid sequence of LCDR3, 006-H07

(63) Seq ID NO:33 represents amino acid sequence of LCDR3, 005-C04

(64) Seq ID NO:34 represents amino acid sequence of VH, 006-H08

(65) Seq ID NO:35 represents amino acid sequence of VH, 002-H06

(66) Seq ID NO:36 represents amino acid sequence of VH, 002-H08

(67) Seq ID NO:37 represents amino acid sequence of VH, 006-H07

(68) Seq ID NO:38 represents amino acid sequence of VH, 001-E06

(69) Seq ID NO:39 represents amino acid sequence of VH, 005-C04

(70) Seq ID NO:40 represents amino acid sequence of VL, 006-H08

(71) Seq ID NO:41 represents amino acid sequence of VL, 002-H06

(72) Seq ID NO:42 represents amino acid sequence of VL, 002-H08

(73) Seq ID NO:43 represents amino acid sequence of VL, 006-H07

(74) Seq ID NO:44 represents amino acid sequence of VL, 001-E06

(75) Seq ID NO:45 represents amino acid sequence of VL, 005-C04

(76) Seq ID NO:46 represents nucleic acid sequence VH, 006-H08

(77) Seq ID NO:47 represents nucleic acid sequence VH, 002-H06

(78) Seq ID NO:48 represents nucleic acid sequence VH, 002-H08

(79) Seq ID NO:49 represents nucleic acid sequence VH, 006-H07

(80) Seq ID NO:50 represents nucleic acid sequence VH, 001-E06

(81) Seq ID NO:51 represents nucleic acid sequence VH, 005-C04

(82) Seq ID NO:52 represents nucleic acid sequence VL, 006-H08

(83) Seq ID NO:53 represents nucleic acid sequence VL, 002-H06

(84) Seq ID NO:54 represents nucleic acid sequence VL, 002-H08

(85) Seq ID NO:55 represents nucleic acid sequence VL, 006-H07

(86) Seq ID NO:56 represents nucleic acid sequence VL, 001-E06

(87) Seq ID NO:57 represents nucleic acid sequence VL, 005-C04

(88) Seq ID NO:58 represents amino acid sequence of VH, HE06642, Novartis (WO2008/22295)

(89) Seq ID NO:59 represents amino acid sequence of VH, XHA06642, Novartis (WO2008/22295)

(90) Seq ID NO:60 represents amino acid sequence of VH, XHA06983, Novartis (WO2008/22295)

(91) Seq ID NO:61 represents amino acid sequence of VL, HE06642

(92) Seq ID NO:62 represents amino acid sequence of VL, XHA06642 Novartis (WO2008/22295)

(93) Seq ID NO:63 represents amino acid sequence of VL, XHA06983 Novartis (WO2008/22295)

(94) Seq ID NO:64 represents nucleic acid sequence VH, HE06642

(95) Seq ID NO:65 represents nucleic acid sequence VH, XHA06642 Novartis (WO2008/22295)

(96) Seq ID NO:66 represents nucleic acid sequence VH, XHA06983 Novartis (WO2008/22295)

(97) Seq ID NO:67 represents nucleic acid sequence VL, HE06642

(98) Seq ID NO:68 represents nucleic acid sequence VL, XHA06642, Novartis (WO2008/22295)

(99) Seq ID NO:69 represents nucleic acid sequence VL, XHA06983, Novartis (WO2008/22295)

(100) Seq ID NO:70 represents human ECD_PRLR, amino acid position 1-210, S1 domain 1-100 (S1 domain construct 1-102), S2 domain 101-210

(101) Seq ID NO:71 represents CDS human ECD_PRLR, nucleotide position 1-630

(102) Seq ID NO:72 represents murine ECD_PRLR, amino acid position 1-210

(103) Seq ID NO:73 represents CDS murine ECD_PRLR, nucleotide position 1-630

(104) SEQ ID NO:74: represents HCDR2, maturated 006-H08 variants, amino acid sequence

(105) SEQ ID NO:75 represents HCDR2, maturated 006-H08 variants, amino acid sequence

(106) SEQ ID NO:76 represents HCDR2, maturated 006-H08 variants, amino acid sequence

(107) SEQ ID NO:77 represents HCDR2, maturated 006-H08 variants, amino acid sequence

(108) SEQ ID NO:78 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(109) SEQ ID NO:79 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(110) SEQ ID NO:80 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(111) SEQ ID NO:81 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(112) SEQ ID NO:82 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(113) SEQ ID NO:83 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(114) SEQ ID NO:84 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(115) SEQ ID NO:85 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(116) SEQ ID NO:86 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(117) SEQ ID NO:87 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(118) SEQ ID NO:88 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(119) SEQ ID NO:89 represents LCDR1, maturated 006-H08 variants, amino acid sequence

(120) SEQ ID NO:90 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(121) SEQ ID NO:91 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(122) SEQ ID NO:92 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(123) SEQ ID NO:93 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(124) SEQ ID NO:94 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(125) SEQ ID NO:95 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(126) SEQ ID NO:96 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(127) SEQ ID NO:97 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(128) SEQ ID NO:98 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(129) SEQ ID NO:99 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(130) SEQ ID NO:100 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(131) SEQ ID NO:101 represents LCDR3, maturated 006-H08 variants, amino acid sequence

(132) SEQ ID NO:143 represents VH, 006-H08-12-2, amino acid sequence

(133) SEQ ID NO:144 represents VH, 006-H08-13-2, amino acid sequence

(134) SEQ ID NO:145 represents VH, 006-H08-13-6-1, amino acid sequence

(135) SEQ ID NO:146 represents VH, 006-H08-14-6-0, amino acid sequence

(136) SEQ ID NO:147 represents VH, 006-H08-15-5, amino acid sequence

(137) SEQ ID NO:148 represents VH, 006-H08-19-1, amino acid sequence

(138) SEQ ID NO:149 represents VH, 006-H08-29-1, amino acid sequence

(139) SEQ ID NO:150 represents VH, 006-H08-32-2, amino acid sequence

(140) SEQ ID NO:151 represents VH, 006-H08-33-0, amino acid sequence

(141) SEQ ID NO:152 represents VH, 006-H08-33-16-0, amino acid sequence

(142) SEQ ID NO:153 represents VH, 006-H08-35-17-1, amino acid sequence

(143) SEQ ID NO:154 represents VH, 006-H08-35-17-4, amino acid sequence

(144) SEQ ID NO:155 represents VH, 006-H08-35-1, amino acid sequence

(145) SEQ ID NO:156 represents VH, 006-H08-36-17-0, amino acid sequence

(146) SEQ ID NO:157 represents VH, 006-H08-37-19-0, amino acid sequence

(147) SEQ ID NO:158 represents VH, 006-H08-39-7, amino acid sequence

(148) SEQ ID NO:159 represents VH, 006-H08-48-5, amino acid sequence

(149) SEQ ID NO:160 represents VH, 006-H08-53-27-0, amino acid sequence

(150) SEQ ID NO:161 represents VH, 006-H08-59-30-0, amino acid sequence

(151) SEQ ID NO:162 represents VH, 006-H08-63-32-4, amino acid sequence

(152) SEQ ID NO:163 represents VH, 006-H08-65-33-2, amino acid sequence

(153) SEQ ID NO:164 represents VH, 006-H08-68-35-2, amino acid sequence

(154) SEQ ID NO:165 represents VL, 006-H08-12-2, amino acid sequence

(155) SEQ ID NO:166 represents VL, 006-H08-13-2, amino acid sequence

(156) SEQ ID NO:167 represents VL, 006-H08-13-6-1, amino acid sequence

(157) SEQ ID NO:168 represents VL, 006-H08-14-6-0, amino acid sequence

(158) SEQ ID NO:169 represents VL, 006-H08-15-5, amino acid sequence

(159) SEQ ID NO:170 represents VL, 006-H08-19-1, amino acid sequence

(160) SEQ ID NO:171 represents VL, 006-H08-29-1, amino acid sequence

(161) SEQ ID NO:172 represents VL, 006-H08-32-2, amino acid sequence

(162) SEQ ID NO:173 represents VL, 006-H08-33-0, amino acid sequence

(163) SEQ ID NO:174 represents VL, 006-H08-33-16-0, amino acid sequence

(164) SEQ ID NO:175 represents VL, 006-H08-35-17-1, amino acid sequence

(165) SEQ ID NO:176 represents VL, 006-H08-35-17-4, amino acid sequence

(166) SEQ ID NO:177 represents VL, 006-H08-35-1, amino acid sequence

(167) SEQ ID NO:178 represents VL, 006-H08-36-17-0, amino acid sequence

(168) SEQ ID NO:179 represents VL, 006-H08-37-19-0, amino acid sequence

(169) SEQ ID NO:180 represents VL, 006-H08-39-7, amino acid sequence

(170) SEQ ID NO:181 represents VL, 006-H08-48-5, amino acid sequence

(171) SEQ ID NO:182 represents VL, 006-H08-53-27-0, amino acid sequence

(172) SEQ ID NO:183 represents VL, 006-H08-59-30-0, amino acid sequence

(173) SEQ ID NO:184 represents VL, 006-H08-63-32-4, amino acid sequence

(174) SEQ ID NO:185 represents VL, 006-H08-65-33-2, amino acid sequence

(175) SEQ ID NO:186 represents VL, 006-H08-68-35-2, amino acid sequence

(176) SEQ ID NO:331 represents VH, 006-H08-12-2, nucleic acid sequence

(177) SEQ ID NO:332 represents VH, 006-H08-13-2, nucleic acid sequence

(178) SEQ ID NO:333 represents VH, 006-H08-13-6-1, nucleic acid sequence

(179) SEQ ID NO:334 represents VH, 006-H08-14-6-0, nucleic acid sequence

(180) SEQ ID NO:335 represents VH, 006-H08-15-5, nucleic acid sequence

(181) SEQ ID NO:336 represents VH, 006-H08-19-1, nucleic acid sequence

(182) SEQ ID NO:337 represents VH, 006-H08-29-1, nucleic acid sequence

(183) SEQ ID NO:338 represents VH, 006-H08-32-2, nucleic acid sequence

(184) SEQ ID NO:339 represents VH, 006-H08-33-0, nucleic acid sequence

(185) SEQ ID NO:340 represents VH, 006-H08-33-16-0, nucleic acid sequence

(186) SEQ ID NO:341 represents VH, 006-H08-35-17-1, nucleic acid sequence

(187) SEQ ID NO:342 represents VH, 006-H08-35-17-4, nucleic acid sequence

(188) SEQ ID NO:343 represents VH, 006-H08-35-1, nucleic acid sequence

(189) SEQ ID NO:344 represents VH, 006-H08-36-17-0, nucleic acid sequence

(190) SEQ ID NO:345 represents VH, 006-H08-37-19-0, nucleic acid sequence

(191) SEQ ID NO:346 represents VH, 006-H08-39-7, nucleic acid sequence

(192) SEQ ID NO:347 represents VH, 006-H08-48-5, nucleic acid sequence

(193) SEQ ID NO:348 represents VH, 006-H08-53-27-0, nucleic acid sequence

(194) SEQ ID NO:349 represents VH, 006-H08-59-30-0, nucleic acid sequence

(195) SEQ ID NO:350 represents VH, 006-H08-63-32-4, nucleic acid sequence

(196) SEQ ID NO:351 represents VH, 006-H08-65-33-2, nucleic acid sequence

(197) SEQ ID NO:352 represents VH, 006-H08-68-35-2, nucleic acid sequence

(198) SEQ ID NO:353 represents VL, 006-H08-12-2, nucleic acid sequence

(199) SEQ ID NO:354 represents VL, 006-H08-13-2, nucleic acid sequence

(200) SEQ ID NO:355 represents VL, 006-H08-13-6-1, nucleic acid sequence

(201) SEQ ID NO:356 represents VL, 006-H08-14-6-0, nucleic acid sequence

(202) SEQ ID NO:357 represents VL, 006-H08-15-5, nucleic acid sequence

(203) SEQ ID NO:358 represents VL, 006-H08-19-1, nucleic acid sequence

(204) SEQ ID NO:359 represents VL, 006-H08-29-1, nucleic acid sequence

(205) SEQ ID NO:360 represents VL, 006-H08-32-2, nucleic acid sequence

(206) SEQ ID NO:361 represents VL, 006-H08-33-0, nucleic acid sequence

(207) SEQ ID NO:362 represents VL, 006-H08-33-16-0, nucleic acid sequence

(208) SEQ ID NO:363 represents VL, 006-H08-35-17-1, nucleic acid sequence

(209) SEQ ID NO:364 represents VL, 006-H08-35-17-4, nucleic acid sequence

(210) SEQ ID NO:365 represents VL, 006-H08-35-1, nucleic acid sequence

(211) SEQ ID NO:366 represents VL, 006-H08-36-17-0, nucleic acid sequence

(212) SEQ ID NO:367 represents VL, 006-H08-37-19-0, nucleic acid sequence

(213) SEQ ID NO:368 represents VL, 006-H08-39-7, nucleic acid sequence

(214) SEQ ID NO:369 represents VL, 006-H08-48-5, nucleic acid sequence

(215) SEQ ID NO:370 represents VL, 006-H08-53-27-0, nucleic acid sequence

(216) SEQ ID NO:371 represents VL, 006-H08-59-30-0, nucleic acid sequence

(217) SEQ ID NO:372 represents VL, 006-H08-63-32-4, nucleic acid sequence

(218) SEQ ID NO:373 represents VL, 006-H08-65-33-2, nucleic acid sequence

(219) SEQ ID NO:374 represents VL, 006-H08-68-35-2, nucleic acid sequence

EXAMPLES

Example 1

Isolation of Target-Specific Antibodies from Human Antibody Phage Display Libraries

(220) To isolate a panel of antibodies able to neutralize the activity of human PRLR, three human antibody phage display libraries, expressing Fab and scFv fragments, were investigated in parallel. The target used for the library panning was the soluble extracellular domain (ECD) of the prolactin receptor represents human prolactin receptor amino acids 25-234, prepared as described above in WO08/022,295 represents (Novartis). Alternative targets were the ECD of PRLR C-terminally linked to six histidines or to a human IgG1-Fc domain via the linker with the amino acid sequence isoleucine-glutamate-glycine-arginine-methionine-aspartate.

(221) Selection of target-specific antibodies from phage display was carried out according to methods described by Marks et al. (Methods Mol. Biol. 248:161-76, 2004). Briefly, the phage display library was incubated with 50 pmols of the biotinylated ECD at room temperature for 1 hr and the complex formed was then captured using 100 l of Streptavidin beads suspension (Dynabeads M-280 Streptavidin, Invitrogen). Non specific phages were removed by washing the beads with wash buffer (PBS+5% Milk,). Bound phages were eluted with 0.5 ml of 100 nM Triethylamine (TEA,) and immediately neutralized by addition of an equal volume of IM TRIS-Cl pH 7.4. Eluted phage pool was used to infect TG1 E. coli cells growing in logarithmic phase, and phagemid was rescued as described (Methods Mol. Biol. 248:161-76, 2004). Selection was repeated for a total of three rounds. Single colonies obtained from TG1 cells infected with eluted phage from the third round of panning were screened for binding activity in an ELISA assay. Briefly, single colonies obtained from the TG1 cell infected with eluted phage were used to inoculate media in 96-well plates.

(222) Microcultures were grown to an OD.sub.60O=0.6 at which point expression of soluble antibody fragment was induced by addition of 1 mM IPTG following overnight culture in a shaker incubator at 30 C. Bacteria were spun down and periplasmic extract was prepared and used to detect antibody binding activity to ECD immobilized on 96-well microplates (96-well flat bottom Immunosorb plates, Nunc) following standard ELISA protocol provided by the microplate manufacturer.

(223) The affinities of the anti-Prolactin Receptor (PRLR) antibodies for binding to the recombinant extracellular domain (ECD) were estimated using the Biacore 2000 and used for affinity ranking of antibodies.

Example 2

Quantitative Analysis of Prolactin and Prolactin Receptor Gene Expression by Real-Time TaqMan PCR Analysis in Eu- and Ectopic Endometrium and Endometriotic Lesions from Patients and Healthy Controls

(224) Real-time Taqman PCR analysis was performed using the ABI Prism 7700 Sequence Detector System according to the manufacturer's instructions (PE Applied Biosystems) and as described Endocrinolgy 2008, 149(8): 3952-3959) and known by the expert in the field. Relative expression levels of PRL and the PRLR were normalized to the expression of cyclophyllin. We analyzed the expression of PRL and the PRLR in the endometrium from healthy women and in endometrium and endometriotic lesions from patients by using quantitative real-time Taqman PCR analysis. The expression of prolactin and its receptor was clearly upregulated in endometriotic lesions compared to healthy endometrium or endometrium derived from patients.

(225) Results are shown in FIGS. 1 and 2.

(226) These findings imply that autocrine prolactin signaling plays a role in the development and maintenance of endometriosis and adenomyosis uteri (endometriosis interna, a form of endometriosis restricted to the uterus.

Example 3

Analysis of Prolactin Receptor Expression in Human Tissues by Northern Blot

(227) RNA was isolated from different rat tissues and transferred to a nylon membrane after gel electrophoresis. The membranes were successively hybridized with radioactive labelled cDNAs for the rat prolactin receptor or -actin (as loading control), washed, and exposed to film. The bands correspond to the mRNAs for the rat prolactin receptor and -actin. The results shown in FIG. 3 indicate a strong expression of the prolactin receptor in the placenta, the prostate, the ovary and the adrenal gland.

Example 4

Regulation of Prolactin Receptor Protein Expression in Rat ProstateInfluence of Castration and Hormonal Treatments

(228) Rats were either castrated or remained intact. Intact animals were treated daily for 14 days with vehicle (intact), DHT (3 mg/kg), or E2 (0.4 mg/kg). Afterwards prostates were isolated from animals of all treatment groups and protein extracts were prepared. Protein extracts were separated by gel electrophoresis and transferred to a membrane. The prolactin receptor was detected using the commercially available antibody MA610 (Santa Cruz Biotechnology). The results are shown in FIG. 4 and indicate the hormonal regulation of the prolactin receptor in the rat prostate.

Example 5

Inhibition of Prolactin-Induced Proliferation of BaF3 Cells (Stably Transfected with Human Prolactin Receptor), by Neutralizing Prolactin Receptor Antibodies and Unspecific Control Antibodies

(229) To analyze the in vitro efficacy of the neutralizing PRLR antibodies, 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 PRLR antibodies 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 IC.sub.50 values calculated. As negative control, stimulation with an unspecific control antibody was used.

(230) The dose-response curves and IC.sub.50 values are depicted in FIG. 5. The unspecific antibody did not inhibit the proliferation of BaF cells stably expressing the human PRLR, whereas the specific antibodies blocked cell proliferation and exhibited different potencies. Neutralizing antibody 006-H08 showed the highest potency in this readout paradigm.

Example 6

Inhibition of Prolactin-Induced Rat Lymphoma Cell Proliferation by Specific and Unspecific Antibodies

(231) The in vitro efficacy of the neutralizing PRLR antibodies was also tested using inhibition of prolactin-dependent rat lymphoma cell (Nb2-11 cells), proliferation. Nb2-11 cells were routinely grown in RPMI containing 10% FCS and 10% horse serum. Before starting cellular growth assays, cells were grown for 24 hours in the same medium containing 1% FCS instead of 10% FCS. Afterwards, cells were seeded in 96-well plates in FCS-free medium at a density of 10000 cells per well. Cells were stimulated with 10 ng/ml human prolactin in the presence or absence of increasing doses of neutralizing PRLR antibodies or control antibodies for 2 days. Afterwards cellular proliferation was assessed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega). Dose-response curves and IC.sub.50 values are depicted in FIG. 6. The unspecific antibody and antibody XHA06.983, that does not bind the rat PRLR, did not block Nb2-11 cell proliferation. XHA06.642 which binds the rat PRLR blocked Nb2-11 cell proliferation.

Example 7

Inhibition of Prolactin-Induced STAT5 Phosphorylation in T47D Cells by Neutralizing Prolactin Receptor Antibodies

(232) To analyze the in vitro efficacy of the neutralizing PRLR antibodies in an additional readout, the inhibition of STAT5 phosphorylation in human T47D cells treated with prolactin was used. T47D cells were grown in RPMI containing 10% FCS and 2 mM glutamine. Cells were seeded on 24-well plates at a density of 0.510.sup.5 cells per well. The next day, cells were starved for 1 h in serum free RPMI. Afterwards cells were incubated with or without different doses of neutralizing PRLR antibodies or unspecific control antibody in the absence or presence of 20 ng/ml human prolactin for 30 min. Afterwards cells were rinsed and lysed in 70 l of lysisbuffer. Lysates were centrifuged and the supernatant was frozen at 80 C. Extracts were analyzed using Western blot (anti-pSTAT5A/B antibody from upstate 07-586, 1:1000 diluted). As loading control the stripped blots were incubated with anti-beta tubulin antibody (ab7287, 1:500 diluted). Results are shown in FIG. 7. With the exception of the unspecific FITC antibody, all neutralizing PRLR antibodies blocked STAT5 phosphorylation in human T47D cells dose-dependently. All tested antibodies bound to the human PRLR with high affinity.

Example 8

Inhibition of Luciferase Reporter Gene Activity in Hek293 Cells Stably Transfected with the Human PRLRAnalysis of Neutralizing Prolactin Receptor Antibodies and Unspecific Control Antibodies

(233) To further analyze the in vitro efficacy of the neutralizing PRLR antibodies, a reporter gene assay was used. HEK293HEK293 cells stably transfected with the human PRLR were transiently transfected with a luciferase reporter gene under the control of LHREs (lactogenic hormone response elements), for 7 hours. Afterwards, cells were seeded at a density of 20000 cells per well on a 96-well plate (0.5% charcoal stripped serum, DMEM). The next day 300 ng/ml human prolactin with and without increasing doses of neutralizing PRLR antibodies or control antibodies was added. 24 hours later, luciferase activity was determined. Results are depicted in FIG. 8. In contrast to the unspecific antibody, 006-H08 and HE06.642 inhibited luciferase activity in HEK293 cells stably transfected with the human PRLR.

Example 9

Inhibition of Luciferase Reporter Gene Activity in Hek293 Cells Stably Transfected with the Murine PRLRAnalysis of Neutralizing Prolactin Receptor Antibodies and Unspecific Control Antibodies

(234) To further analyze the in vitro efficacy of the neutralizing PRLR antibodies on the murine prolactin receptor, a reporter gene assay was used. HEK293 cells stably transfected with the murine PRLR were transiently transfected with a luciferase reporter gene under the control of LHREs (lactogenic hormone response elements) for 7 hours. Afterwards, cells were seeded at a density of 20000 cells per well on a 96-well plate (0.5% charcoal stripped serum, DMEM). The next day 200 ng/ml human prolactin with and without increasing doses of neutralizing PRLR antibodies or control antibodies was added. 24 hours later, luciferase activity was determined. Results are depicted in FIG. 9. Whereas the antibody 005-C04 (closed triangles) exhibits high activity (IC.sub.50 value=3 nM), the antibody HE06.642 (closed circles) does not show activity up to 330 nM. The unspecific control antibody (open circles) is completely inactive. In contrast to the Novartis antibody HE06.642, the antibody 005-C04 is able to block murine PRLR-mediated signaling.

Example 10

Inhibition of Prolactin-Induced Proliferation of BaF3 Cells (Stably Transfected with the Murine Prolactin Receptor) by Neutralizing Prolactin Receptor Antibodies and Unspecific Control Antibodies

(235) To analyze the in vitro efficacy of the neutralizing PRLR antibodies, the inhibition of prolactin-activated cellular proliferation of Ba/F3 cells was used. The cells were stably transfected with the murine 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 40 ng/ml prolactin and coincubated with increasing doses of neutralizing PRLR antibodies 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 IC.sub.50 values calculated. As negative control, stimulation with an unspecific control antibody was used.

(236) The dose-response curves and IC.sub.50 values are depicted in FIG. 10. The unspecific control antibody (closed squares) was inactive at the murine PRLR. There was only limited inhibition of murine PRLR activation by the antibodies HE06.642, 001-E06, and 001-D07. Only antibody 005-C04 completely blocked murine PRLR activation.

Example 11

Contraceptive Effect of Neutralizing Prolactin Receptor Antibody IgG1 005-C04 in Mice

(237) To test the influence of neutralizing prolactin receptor antibodies on fertility in mice, 12 week old female and male NMRI mice were mated for 7 days (day 0-day 7). Female mice were treated on days 3, 0, 3, and 6 with an intraperitoneal injection of either phosphate-buffered saline, unspecific IgG1 control antibody (anti-FITC, 10 mg/kg), or the neutralizing IgG1 antibody 005-C04 (=IgG1 005-C04) at concentrations of 10 or 30 mg per kg body weight dissolved in phosphate buffered saline. 10 females were used in each experimental group. Each male was mated with two females, one of the females was from a negative control group treated with either phosphate-buffered saline or unspecific antibody, the other female was treated with specific neutralizing antibody. Matings, in which the male did not produce at least one pregnant female, were excluded from data evaluation. Readout parameters were mean litter size and pregnancy rates (measured in %) calculated as litter number per experimental group divided by the number of theoretical possible litters within this group. Results are depicted in FIG. 11.

(238) FIG. 11A shows the obtained pregnancy rates. Pregnancy rates were as follows: 87.5% in the group of mice treated with phosphate buffered saline, 75% in the group of mice treated with the unspecific control antibody (10 mg/kg), 100% in the group of mice treated with the neutralizing PRLR antibody IgG1 005-C04 (10 mg/kg), and 0% in the group of mice treated with the neutralizing PRLR antibody IgG1 005-C04 (30 mg/kg).

(239) FIG. 11B shows the observed litter sizes for the different experimental groups. Litter sizes were as follows: 10.9 mice per litter in the group of mice treated with phosphate buffered saline, 12.3 mice per litter in the group of mice treated with the unspecific control antibody (10 mg/kg), 13 mice per litter in the group of mice treated with the neutralizing PRLR antibody IgG1 005-C04 (10 mg/kg), and 0 mice per litter in the group of mice treated with the neutralizing PRLR antibody IgG1 005-C04 (30 mg/kg).

(240) The results from this mating study demonstrate that the neutralizing prolactin receptor antibody IgG1-005-C04 completely prevented pregnancy in mice when tested at 30 mg/kg body weight.

Example 12

Epitope Grouping

(241) Epitope grouping experiments were performed using Biacore by monitoring simultaneous binding of pairs of anti-PRLR antibodies to ECD-PRLR (SEQ ID NO: 70).

(242) Briefly, the first antibody was covalently immobilized to the sensor chip through primary amine coupling using n-hydroxysuccinamide (NHC) and N-ethyl-N-dimethylaminopropyl carbodiimide (EDC). Unoccupied binding sites on the surface were then blocked with ethanolamide. Soluble ECD-PRLR (SEQ ID NO: 70) was captured on the surface via the immobilized antibody, therefore, the epitope of the capture antibody is blocked for all bound ECD-PRLR molecules. A second antibody was immediately passed over the surface to bind to the immobilized ECD-PRLR. Two antibodies recognizing the same or overlapping epitopes cannot bind to the ECD-PRLR, whereas antibodies with distinct epitopes are able to bind. The antibody surface was regenerated with glycine, pH 2.8, to remove bound proteins and then the process was repeated with other antibodies. All combinations of antibodies were tested. Representative results are shown in Table 7. The antibodies 006-H08, 002-H06, 002-H08, 006-H07 and XHA06983 competitively bound to each other on ECD-PRLR, indicating that they target overlapping epitopes (epitope group 1, table 6). In addition, the antibodies competitively bound to PRL, which is also the case for 001-E06 (epitope group 2, table 6). This antibody targets a different site of ECD-PRLR than the afore mentioned ones. Finally, the antibody 005-C04 competitively bound to HE06.642 and XHA06.642 without being competitive to PRL (epitope group 3, table 6).

(243) TABLE-US-00007 TABLE 7 Groups of antibodies which target overlapping epitopes on the extracellular domain (ECD) of the human prolactin receptor (PRLR) Competition to Antibody Epitope group prolactin 006-H08 1 Yes 002-H06 1 Yes 002-H08 1 Yes 006-H07 1 Yes 001-E06 2 Yes 005-C04 3 No HE06.642 3 No XHA06.642 3 No XHA06.983 1 Yes

Example 13

Cross-Reactivity of Antibodies on Mouse And Human PRLR Expressed on Cell Surfaces

(244) In order to determine the binding characteristics of the anti-PRLR antibodies on mouse and human PRLR expressed on cells, binding was tested by flow cytometry on HEK293 cells stably expressing the human and murine PRLR, respectively. The cells as well as the parental HEK293 cell line without PRLR were harvested, centrifuged and resuspended at approximately 510.sup.6 cells/ml in 1PBS containing 2% FBS and 0.1% sodium azide (FACS buffer). The antibodies 005-C04, 001-E06 and HE06.642 were diluted to 2-fold final concentration in FACS buffer and added to appropriate sample wells (50 l/well). For secondary antibody and autofluorescence controls, 50 l FACS buffer was added to appropriate wells. 50 l of cell suspension was added to each sample well. Samples were incubated at 4 C. for one hour, washed twice with cold FACS buffer and resuspended in FACS buffer containing PE-conjugated goat anti-human IgG at a 1:100 dilution. Following a 30 min incubation at 4 C., cells were washed twice with cold FACS buffer, resuspended in FACS buffer containing 1 mg/ml propidium iodide (Invitrogen, San Diego, Calif.) and analyzed by flow cytometry. As shown in FIG. 13, the antibodies 005-C04 and 001-E06 bound to human and murine PRLR on these cells, while HE06.642 only bound to the human PRLR. This observation is consistent with the finding reported in example 9 about the missing efficacy of HE06.642 in the murine PRLR-dependent luciferase reporter gene assay. Although 005-C04 and HE06.642 competitively bound to human PRLR, the different binding properties of both antibodies with respect to the murine PRLR indicate differences in their epitope specificity.

Example 14

Inhibitory Activity of Fab and scFv Antibodies on Cellular Signaling Cascades

(245) To functionally characterize the activity of the Fab and scFv screening hits on the PRLR-triggered signaling cascade, the inhibition of phosphorylation on PRLR itself, and on the transcriptional regulators ERK1/2 and STAT5 in human T47D cells treated with prolactin was measured. T47D cells were grown in RPMI containing 2 mM L-glutamine, 10% charcoal stripped FBS and insulin-transferrin-selenium-A (Gibco). Cells were seeded on 6 well plates or 96-well plates at a density of 1.510.sup.6 cells per well. The next day, growth medium was renewed. On the third, day cells were starved for 1 hour in serumfree RPMI. Afterwards cells were incubated with or without different doses of neutralizing PRLR antibodies or unspecific control antibody in the presence of 500 ng/ml human prolactin for 5 min. Afterwards cells were rinsed and lysed in lysis buffer. Lysates were centrifuged and the supernatants were frozen at 80 C. Samples were tested by ELISA according to the DuoSet IC Human Phospho-Prolactin R kit (R&D Systems) for measurement of PRLR phosphorylation, according to the PathScan Phospho-STAT5 (Tyr694) Sandwich ELISA kit (Cell Signaling Technology; #7113) for measurement of STAT5 phosphorylation and according to the Phospho-ERK1/ERK2 kit (R&D Systems) for measurement of ERK1/2 phosphorylation. Table 8 provides an overview about the antagonistic activity of a selection of screening hits in Fab or scFv format at a fixed dose of 7.5 g per ml.

(246) TABLE-US-00008 TABLE 8 Antagonistic activity of a selection of screening hits on the phosphorylation of PRLR, ERK1/2 and STAT5 as determined by ELISAs on cell lysates of the human breast cancer cell line T47D Inhibition of phosphorylation in % at a fixed antibody dose (7.5 g/ml) Antibody PRLR ERK1/2 STAT5 006-H08* 100 100 100 002-H06.sup. 92 86 72 002-H08.sup. 100 100 98 006-H07* 88 85 73 001-E06.sup. 63 45 36 Negative control 2 9 0 *scFv format, Fab format

Example 15

Neutralizing PRLR Antibodies Inhibit Lactation in Mice

(247) Adult NMRI females were mated with NMRI males. On postpartal day 1, litter size was adjusted to 8 mice per lactating mother. The weight of the offspring was determined daily in the morning starting on postpartal day 1. Lactating mothers remained either untreated (closed circles in FIG. 14A,B) or were treated intraperitoneally with either unspecific antibody (10 mg/kg body weight; open circles in FIG. 14A,B), or with neutralizing PRLR antibody 005-C04 containing murine IgG2a constant domains (=IgG2a 005-C04; 10 mg/kg, closed triangles in FIG. 14A, B) or with neutralizing PRLR antibody IgG2a 005-C04 (30 mg/kg, open triangles in FIG. 14A, B). Group size was 5-6 lactating mothers per experimental group. Mothers were treated with specific or unspecific control antibodies on postpartal day 1, 3, 6, 9, 10, and 12 (indicated with arrows in FIG. 14A, B). The results are depicted in FIG. 14. FIG. 14A shows for each postpartal day the daily litter weight gain expressed as percentage of the respective litter weight on day 1. From postpartal day 8 onwards there is a significant difference in litter weight gain between offspring from mothers treated with neutralizing PRLR antibodies and offspring from mothers that remained untreated or received unspecific control antibodies. Due to ethical reasons several litters had to be killed on postpartal day 10 in the experimental group of mothers receiving the highest dose of the neutralizing PRLR antibody. In FIG. 14B the results are depicted in a different way. The differential litter weight gain from day to day is depicted and expressed as percentage of the litter weight on postpartal day 1. Basically FIG. 14B shows the slope of the graphs depicted in FIG. 14A. The differential daily increase in litter weight oscillates around 30% of the starting litter weight on postpartal day 1 for litters from untreated mothers or mothers treated with the unspecific antibody. There is a significant severe reduction in daily litter weight increase in litters from mothers treated with the neutralizing PRLR antibody at 30 mg/kg body weight from day 7 onwards (*p<0.05; ***p<0.005 vs. litters from mothers treated with unspecific antibody). From postpartal day 11 onwards, daily litter weight increase is significantly diminished also in litters from mothers treated with the neutralizing PRLR antibody at 10 mg/kg if compared to litters from mothers treated with unspecific control antibodies (p<0.05 vs. litters from mothers treated with unspecific antibody). In conclusion, there are dose-dependent effects of the neutralizing PRLR antibody IgG2a 005-C04 on lactation inhibition. FIG. 14C shows histological sections of the mammary glands from lactating mothers of the different experimental groups. Mammary glands of untreated mothers and mothers treated with unspecific control antibodies are filled with ducts producing milk. In contrast, there are signs of mammary gland involution in mothers treated with the neutralizing PRLR antibody IgG2a 005-C04. Black arrows in FIG. 14C point to fatty islands in the mammary gland tissue (see dose-dependent effect of the specific antibody IgG2a 005-C04 on the extent of mammary gland involution (FIG. 14C)). In addition, the expression of the major milk proteins beta-casein (Csn-2), whey acidic protein (WAP), and IGF-1 in the mammary glands of mothers from the different experimental groups were analyzed (FIG. 14D). Gene expression was normalized to the expression of TATA-box binding protein (TBP). The neutralizing PRLR antibody IgG2a 005-C04 dose-dependently decreased milk protein expression whereas the unspecific antibody (10 mg/kg) was without any significant effect.

(248) The neutralizing PRLR antibody IgG2a 005-C04 dose-dependently blocked lactation and lead to mammary gland involution in lactating mice demonstrating its usefulness for lactation inhibition.

Example 16

Neutralizing PRLR Antibodies are Suitable for the Treatment of Benign Breast Disease

(249) An activating PRLR mutation or local or systemic hyperprolactinemia can provoke benign breast disease. Therefore, a hyperprolactinemic mouse model to induce enhanced proliferation in the mammary gland (hallmark of the most severe forms of benign breast disease) was employed. On day 0, 12 week old female Balb/c mice received a pituitary isograft under the kidney capsule or remained unoperated. Pituitary isografted mice remained untreated or were treated intraperitoneally with either unspecific antibody (10 mg/kg), neutralizing PRLR antibody 005-C04 in IgG1 format (=IgG1 005-C04; 10 mg/kg), or neutralizing PRLR antibody IgG1 005-C04 (30 mg/kg) on day 0, 3, 7, 11, and 15. Experimental group size was 8-10 animals. On day 17 after pituitary transplantation mice were sacrificed. Two hours before death, animals received an intraperitoneal injection of BrdU to monitor epithelial cell proliferation. The left inguinal mammary gland was fixed in Carnoy's solution and mammary gland whole mounts were prepared and stained with Carmine alaune (FIG. 15A). The right inguinal mammary gland was fixed in 4% phosphate-buffered formaline overnight. Mammary glands were subsequently embedded in paraffin and BrdU immunostainings were performed as described previously (Endocrinology 149(8):3952-3959; 2009). In addition, a pSTAT5 immunostaining was performed (anti pSTAT5 antibody from abcam, ab32364, diluted 1:60) to monitor the inhibition of PRLR-mediated signaling in response to treatment with neutralizing PRLR antibodies. FIG. 15A shows magnifications of mammary gland whole mounts from the different experimental groups. Mammary glands of adult mice that did not receive a pituitary show ducts and endbuds, whereas there is extreme side branching and formation of alveolar structures in mice receiving a pituitary isograft. Treatment with the unspecific antibody (10 mg/kg) did not inhibit side branching and formation of alveolar structures. In contrast, treatment with the neutralizing antibody IgG1 005-C04 at 10 mg/kg body weight leads to complete inhibition of side branching in 8 out of 10 animals receiving a pituitary isograft and treatment with IgG1 005-C04 at 30 mg/kg completely inhibits side branching in 9 out of 9 animals receiving a pituitary isograft. Histological analysis and BrdU immunostaining are depicted in FIG. 15B. Pituitary isografting leads to epithelial hyperplasia that is not inhibited by treatment with the unspecific antibody, whereas there is no epithelial hyperplasia in mice harbouring a pituitary isograft and treated with the neutralizing PRLR antibody at a dose of 10 or 30 mg/kg body weight. Some of the BrdU-positive cells, reflecting cells in the S-phase of the cell cylcle which are going to divide, are indicated by white arrows in FIG. 15B. Mice treated with the neutralizing antibody IgG1 005-C04 (30 mg/kg body weight) showed almost complete inhibition of epithelial cell proliferation in mammary glands. Some of the cells positive for phospho-STAT5 are indicated by white arrows in FIG. 15C. Treatment with 30 mg/kg IgG1 005-C04 lead to complete inhibition of STAT5 phosphorylation, indicating complete blockade of PRLR-mediated signaling.

(250) The results from FIGS. 15A, B, and C demonstrated that neutralizing PRLR antibodies are suitable for the treatment of mastopathia, a benign proliferative disease of the mammary gland. Neutralizing PRLR antibodies inhibit mammary epithelial cell proliferation and activation of phospho-STAT5.

Example 17

Treatment of Benign Prostate Hyperplasia with Neutralizing PRLR Antibodies

(251) Benign prostate hyperplasia was established in male Balb/c mice by grafting of two pituitaries under the kidney capsule at the age of 8 weeks. A control group remained unoperated. Mice receiving pituitary isografts remained untreated or received intraperitoneal injections of either an unspecific antibody (10 mg/kg), or the neutralizing PRLR antibody 005-C04 containing murine IgG2a constant domains (=IgG2a 005-C04) at doses of 10 and 30 mg/kg body weight. Antibody injections were performed starting on the day of pituitary transplantation (=day 0), and on day 3, day 7, day 11, day 15, day 18, day 22, and day 25 after pituitary transplantation. Mice were sacrificed on day 28. The relative weight of the ventral prostate was determined. Results are depicted in FIG. 16. Pituitary isografting resulted in an increase in relative prostate weight. Treatment with 10 mg/kg and 30 mg/kg neutralizing PRLR antibody IgG2a 005-C04 reduced prostate weight whereas treatment with unspecific control antibody was without any effect. Neutralizing PRLR antibodies are therefore suitable for the treatment of benign prostate hyperplasia.

(252) On day 18 after pituitary isografting it became evident that hair growth was diminished in animals receiving pituitary isografts. Neutralizing PRLR anibodies stimulated hair growth under hyperprolactinemic conditions. Representative photographs are shown in FIG. 17. Therefore neutralizing PRLR antibodies can be used for the treatment of hyperprolactinemic hair loss.

Example 18

Effect of Neutralizing PRLR Antibodies on Hair Growth

(253) The dorsal hair of 8 weeks old male and female C57BL/6 mice was removed using electric shavers as described previously (British Journal of Dermatology 2008; 159:300-305). Hyperprolactinemia was induced in some groups by pituitary isografting under the kidney capsule, animals in the remaining groups were normoprolactinemic. Animals were treated with specific PRLR antibodies (IgG2a 005-C04) or unspecific control antibodies (30 mg/kg, intraperitoneally) once weekly (starting on day 0 which is the day of pituitary isografting). Subsequent antibody injections were performed on days 7 and 14. After three weeks, the regrown hair was visible as dark on the pinkish-white shaved skin, and the percentage of the shaved area that became dark was measured. Female mice were killed 15 days after shaving and male mice were sacrificed 18 days after shaving.

(254) The following experimental groups were used (group size was 6 mice): 1. shaved females 2. shaved females with pituitary isograft 3. shaved females with pituitary isograft+30 mg/kg unspecific antibody IgG2a 005-C04 once weekly 4. shaved females with pituitary isograft+30 mg/kg specific antibody once weekly 5. shaved females+30 mg/kg unspecific antibody once weekly 6. shaved females+30 mg/kg specific antibody once weekly 7. shaved males 8. shaved males with pituitary isograft 9. shaved males with pituitary isograft+30 mg/kg unspecific antibody once weekly 10. shaved males with pituitary isograft+30 mg/kg specific antibody IgG2a 005-C04 once weekly 11. shaved males+30 mg/kg unspecific antibody once weekly 12. shaved males+30 mg/kg specific antibody once weekly

(255) Representative pictures from animals of the different groups are depicted in FIG. 18, the percentage of the area regrown with hair is indicated in FIG. 18.

(256) Neutralising PRLR antibodies, but not unspecific antibodies, stimulate hair regrowth under hyper- and normoprolactinemic conditions in male and female mice. Neutralising PRLR antibodies are therefore suitable to treat hair loss in women and men under hyper- and normoprolactinemic conditions.

Example 19

Inhibition of Enhanced Mammary Epithelial Cell Proliferation by Neutralizing PRLR Antibodies

(257) To test the effect of neutralizing PRLR antibodies on enhanced mammary epithelial cell proliferation activated by combined hormone therapy (i.e. estrogen plus progestin therapy) a previously described mouse model that allowed for the quantification of proliferative effects in the uterus and the mammary gland was employed (Endocrinology 149:3952-3959, 2008). 6 week old C57BL/6 female mice were ovariectomized. 2 weeks after ovariectomy, animals were treated subcutaneously with daily injections of either vehicle (ethanol/arachisoil 10%/90%) or 100 ng estradiol plus 100 mg/kg progesterone for two weeks. Animals were treated once weekly with intraperitoneal injections of neutralizing PRLR antibodies (10 mg/kg and 30 mg/kg) in the murine IgG2a format or unspecific antibody (30 mg/kg) for three weeks. Autopsy was performed on day 36 after ovariectomy. Two hours before death animals received an intraperitoneal injection of bromodeoxyuridine (BrdU) dissolved in phosphate buffered saline (70 mg/kg body weight). The proximal 2/3 of the right inguinal mammary gland was analyzed for mammary epithelial cell proliferation (BrdU immunostaining) described previously (Endocrinology 149:3952-3959, 2008).

(258) The experiment comprised the following groups: 1. ovariectomized animals treated with vehicle 2. ovariectomized animals treated with 100 ng estradiol 3. ovariectomized animals treated with 100 ng estradiol (E) and 100 mg/kg progesterone (P) 4. ovariectomized animals treated with E+P and 10 mg/kg specific antibody 005-C04 5. ovariectomized animals treated with E+P and 30 mg/kg specific antibody 005-C04 6. ovariectomized animals treated with E2+P and 30 mg/kg unspecific control antibody

(259) The results are shown in FIG. 19. The absolute number of proliferating ductal epithelial cells within 4 cross-sections of the mammary gland was evaluated. The medians are depicted as horizontal bars. Epithelial cell proliferation in ovariectomized, vehicle treated mice is rather low. Estradiol treatment leads to some stimulation of epithelial cell proliferation, maximal mammary epithelial cell proliferation is observed under estrogen plus progesterone treatment (FIG. 19). Treatment with neutralising prolactin receptor antibody 005-C04 but not with unspecific control antibody leads to a dose-dependent decrease in mammary epithelial cell proliferation almost back to estradiol-only levels.

(260) Neutralising PRLR antibodies are therefore suitable to treat enhanced mammary epithelial cell proliferation under combined hormone therapy, i.e. estradiol plus progesterone treatment.

Example 20

Treatment of Adenomyosis Uteri (=Endometriosis Interna) in SHN Mice with Neutralizing PRLR Antibodies

(261) To test the efficacy of neutralizing PRLR antibodies in endometriosis, the adenomyosis uteri model in SHN mice relying on systemic hyperprolactinemia was employed (Acta anat. 116:46-54, 1983). Hyperprolactinemia in SHN mice was induced by pituitary isografting under the kidney capsule of 7 weeks old female mice (Acta anat. 116:46-54, 1983). Neutralizing PRLR antibodies (10 mg/kg or 30 mg/kg) or unspecific antibodies (30 mg/kg) were administered intraperitoneally starting one week after pituitary isografting. The infiltration of the uterine muscular layer by glandular tissue was assessed as described previously (Laboratory Animal Science 1998, 48:64-68). Treatment with the antibodies was performed for 9 weeks once and twice weekly by intraperitoneal injections. At autopsy (day 70 after pituitary transplantation), uteri were fixed overnight in buffered 4% formalin and embedded in paraffin. The degree of adenomyosis (=endometriosis interna) was assessed as follows: Grade 0=no adenomyosis Grade 0.5=the inner layer of the myometrium looses its concentric orientation Grade 1=endometrial glands invading the inner layer of the myometrium Grade 2=endometrial glands between the inner and outer layer of the uterine myometrium Grade 3=endometrial glands invading the outer layer of the uterine myometrium Grade 4=endometrial glands outside of the outer layer of the uterine myometrium

(262) The experiment comprised the following experimental groups: 1. Animals without pituitary transplantation, i.e. normoprolactinemic mice 2. Animals with pituitary transplantation, i.e. hyperprolactinemic mice 3. Animals with pituitary transplantation, treated with unspecific control antibody once weekly at a dose of 30 mg/kg 4. Animals with pituitary transplantation, treated with unspecific control antibody twice weekly at a dose of 30 mg/kg 5. Animals with pituitary transplantation, treated with the neutralizing prolactin receptor antibody 005-C04 in the murine IgG2a format once weekly at a dose of 10 mg/kg 6. Animals with pituitary transplantation, treated with the neutralizing prolactin receptor antibody 005-C04 in the murine IgG2a format twice weekly at a dose of 10 mg/kg 7. Animals with pituitary transplantation, treated with the neutralizing prolactin receptor antibody 005-C04 in the murine IgG2a format once weekly at a dose of 30 mg/kg 8. Animals with pituitary transplantation, treated with the neutralizing prolactin receptor antibody 005-C04 in the murine IgG2a format twice weekly at a dose of 30 mg/kg

(263) The results are depicted in FIG. 20. The scores for each animal in each treatment group are given individually and the medians for each treatment group are shown as horizontal bars. Normoprolactinemic mice develop endometriosis interna to some degree (median disease score=0.25). Hyperprolactinemia due to pituitary isografting enhances the disease score and more animals suffer from the disease (median disease score=2.5). Whereas treatment with 30 mg/kg unspecific antibody once or twice weekly had no influence on the disease, treatment with specific neutralizing antibodies shows a dose-dependent decrease in the disease score. Notably, all animals receiving either 10 or 30 mg/kg specific antibody twice weekly were completely cured and their disease score was significantly lower than the disease score of normoprolactinemic mice (FIG. 20). Neutralising PRLR antibodies are therefore suitable to treat endometriosis interna (=adenomyosis uteri) and endometriosis externa in women.

Example 21

Maturation of Antibody Variants

(264) Antibody affinity maturation is a two step process where saturation mutagenesis and well-based high throughput screening are combined to identify a small number of mutations resulting in affinity increases. In the first round of affinity maturation positional diversification of wild-type antibody is introduced by site-directed mutagenesis using NNK-trinucleotide cassettes (whereby N represents a 25% mix each of adenine, thymine, guanine, and cytosine nucleotides and K represents a 50% mix each of thymine and guanine nucleotides) according to BMC Biotechnology 7: 65, 2007. This way, all 20 amino acids are introduced at an individual amino acid position. This positional randomization is restricted to the six complementarity determining regions (CDRs). In the second round of affinity maturation beneficial substitutions were recombined and screened for further improvements.

(265) Screening of Maturated 006-H08 Fab Variants by Homogenous Time-Resolved Fluorescence Assay (HTRF):

(266) Normalized E. coli-derived supernatants and 8 nM biotinylated extracellular domain of PRLR together with 1 nM Streptavidin-Europium were incubated in 96 well-microtiter plates for 30 min. Afterwards 200 mM KF and 50 nM Alexa Fluor 647-labeled IgG1 of 006-H08 were added. This mixture was incubated at room temperature for 5, 20, 35, 50, and 65 minutes, respectively. At the indicated time points, the absorption at 665 nm (and 620 nm) was measured using EnVision MultilabelReader (Perkin Elmer). The obtained results are shown in FIGS. 21A and 21B.