WO2011069795A1 - Neutralizing prolactin receptor antibodies and their therapeutic use - Google Patents

Neutralizing prolactin receptor antibodies and their therapeutic use Download PDF

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Publication number
WO2011069795A1
WO2011069795A1 PCT/EP2010/067742 EP2010067742W WO2011069795A1 WO 2011069795 A1 WO2011069795 A1 WO 2011069795A1 EP 2010067742 W EP2010067742 W EP 2010067742W WO 2011069795 A1 WO2011069795 A1 WO 2011069795A1
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seq
acid sequence
antibody
sequence according
amino acid
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PCT/EP2010/067742
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WO2011069795A4 (en
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Christiane Otto
Siegmund Wolf
Christoph Freiberg
Axel Harrenga
Simone Greven
Mark Trautwein
Sandra Bruder
Andrea Eicker
Andreas Wilmen
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Bayer Schering Pharma Ag
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Priority to RS20170010A priority Critical patent/RS55589B1/sr
Priority to CA2783513A priority patent/CA2783513C/en
Priority to ES10782255.3T priority patent/ES2610654T3/es
Priority to SI201031367A priority patent/SI2510006T1/sl
Priority to AU2010330161A priority patent/AU2010330161B2/en
Priority to EA201200860A priority patent/EA028678B1/ru
Priority to JP2012542432A priority patent/JP2013513361A/ja
Priority to US13/514,992 priority patent/US9649374B2/en
Priority to SG2012041182A priority patent/SG181513A1/en
Priority to IN5082DEN2012 priority patent/IN2012DN05082A/en
Priority to NZ600511A priority patent/NZ600511A/en
Priority to KR1020127017831A priority patent/KR101765968B1/ko
Priority to EP10782255.3A priority patent/EP2510006B1/de
Priority to LTEP10782255.3T priority patent/LT2510006T/lt
Application filed by Bayer Schering Pharma Ag filed Critical Bayer Schering Pharma Ag
Priority to MX2012006621A priority patent/MX2012006621A/es
Priority to DK10782255.3T priority patent/DK2510006T3/en
Priority to BR112012015852A priority patent/BR112012015852B8/pt
Priority to CN201080063269.4A priority patent/CN102884082B/zh
Publication of WO2011069795A1 publication Critical patent/WO2011069795A1/en
Publication of WO2011069795A4 publication Critical patent/WO2011069795A4/en
Priority to IL220149A priority patent/IL220149A/en
Priority to CU2012000092A priority patent/CU20120092A7/es
Priority to ZA2012/04214A priority patent/ZA201204214B/en
Priority to HK13108063.0A priority patent/HK1180700A1/xx
Priority to HRP20170016TT priority patent/HRP20170016T1/hr
Priority to CY20171100030T priority patent/CY1118407T1/el

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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention is directed towards the prolactin receptor antibody 006-H08 and provides recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions, that specifically bind and neutralize the prolactin receptor, nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions containing them and their use in the treatment or prevention of benign diseases and indications which benefit from inhibition of prolactin receptor mediated signaling such as endometriosis, adenomyosis, non- hormonal female contraception, benign breast disease, mastalgia, lactation inhibition, benign prostate hyperplasia, fibroids as well as hyper- and normoprolactinemic hair loss, and cotreatment in combined hormone therapy to inhibit mammary epithelial cell proliferation.
  • prolactin receptor mediated signaling such as endometriosis, adenomyosis, non- hormonal female contraception, benign breast disease, mastalgia, lactation inhibition, benign prostate hyperp
  • Prolactin 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.
  • GH growth hormone
  • PL placental lactogen
  • 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).
  • PRLR PRL receptor
  • 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.
  • PRLR activation 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.
  • JAK/STAT Janus Kinase/Signal transducers and activators of transcription
  • JAKs Upon receptor dimerization, JAKs (predominantly JAK2) associated with the receptor, transphosphorylate and activate each other.
  • 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.
  • STATs SH2-domain containing proteins
  • Receptor bound STATs are subsequently phosphorylated, dissociate from the receptor and translocate to the nucleus where they stimulate transcription of target genes.
  • 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 signaling 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).
  • 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.
  • the problem is solved by provision of the antibody 006-H08, and antigen- binding fragments thereof, or variants thereof for the treatment of the afore mentioned benign diseases and indications, that bind to PRLR with high affinity, efficiently neutralize the PRLR-mediated signaling, and that are preferably cross-reactive to PRLR from other species such as Macacca mulatta and Macacca fascicularis, Mus musculus or Rattus norvegicus.
  • PRLR antibodies have already been described in the application WO2008/022295 (Novartis) and in the US Patent 7,422,899 (Biogen).
  • the present invention is based on the discovery of novel antibodies that 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 (seqences of novel antibodies are as in SEQ ID NO: 34, 40, 46, and 52).
  • the antibodies of the invention which may be human or humanized or chimeric or human engineered, can be used in many contexts which are more fully described herein.
  • an object of the present invention is an antibody or antigen-binding fragment, whereby said antibody antagonizes prolactin receptor-mediated signaling.
  • novel antibodies '002-H06', '002-H08', '006-H07', '001 -E06', '005-C04' are subject matter of corresponding applications.
  • Proliferation assays were performed with rat Nb2-1 1 cells (Example 6, Figure 6) or Ba/F cells either stably transfected with the human PRLR (Example 5, Figure 5) or the murine PRLR (Example 10, Figure 10).
  • Novartis antibody XHA 06.983 did not show activity on the rat and murine PRLR
  • Novartis antibody XHA06.642 showed activity on the rat PRLR but not on the murine PRLR.
  • XHA 06.642 inhibited human PRLR-mediated signaling (Example 5, 7, 8).
  • the novel antibody of the present invention 006-H08 showed the highest potency with regard to proliferation inhibition of Ba/F cells stably transfected with the human PRLR (Example 5, Figure 5).
  • the novel antibody 005-C04 of a corresponding application was the only antibody showing crossreactivity on the murine (Example 10, 9) and human PRLR (Examples 5, 7, 8). In contrast to the Novartis antibody XHA06.642 the novel antibody 005-C04 is therefore suitable for testing the inhibition of PRLR-mediated signaling in murine models. All other antibodies described in this application or in the corresponding applications are specific for the human PRLR.
  • luciferase reporter assays were performed using HEK293 cells stably transfected with either the human (Example 8) or murine (Example 9) PRLR and transiently transfected with a luciferase reporter gene under the control of LHRE's (lactogenic hormone response elements).
  • LHRE's lactogenic hormone response elements
  • the present invention relates to methods to inhibit growth of PRLR-positive cells and the progression of the afore mentioned benign diseases and indications by providing anti-PRLR antibodies.
  • Provided are human monoclonal antibodies, antigen-binding fragments thereof, and variants of the antibodies and fragments, that specifically bind to the extracellular domain (ECD) of PRLR (SEQ ID NO: 70) or human polymorphic variants of SEQ ID No: 70 such as the I 146L and I76V variants being described in PNAS 105 (38), 14533, 2008, and J. Clin. Endocrinol. Metab. 95 (1 ), 271 , 2010.
  • Another object of the present invention is an antibody which binds to epitopes of the extracellular domain of the prolactin receptor and human polymorphic variants thereof, whereby the amino acid sequence of the extracellular domain of the prolactin receptor corresponds to SEQ ID NO: 70, and the nucleic acid sequence corresponds to SEQ ID NO: 71.
  • the antibodies, antigen-binding fragments, and variants of the antibodies and fragments of the invention are comprised of a light chain variable region and a heavy chain variable region.
  • Variants of the antibodies or antigen-binding fragments contemplated in the invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment for PRLR is maintained (for sequences see table 5).
  • an object of the present invention is an antibody or antigen-binding fragment, whereby the antibody or the antigen-binding fragment competes to the antibody 006- H08 or defined maturated variants thereof.
  • the sequences of the antibodies and its maturate variants are depicted in table 5.
  • an antibody or antigen-binding fragment are disclosed, a. whereby the amino acid sequences of the variable heavy and light regions are at least 60%, more preferred 70%, more preferred 80%, or 90%, or even more preferred 95 % identical to SEQ ID NO: 34 for the variable heavy chain domain and, identical to SEQ ID NO: 40 for the variable light chain domain, or
  • amino acid sequences of the variable heavy chain and light chain domain are at least 60%, more preferred 70%, more preferred 80%, or 90%, or even more preferred 95 % identical thereto, or
  • the amino acid sequences of the CDRs are at least 60%, more preferred 70%, more preferred 80%, more preferred 90%, or even more preferred 95 % identical to SEQ ID NO: 1 , 7, and 13 for the heavy chain domain, and to SEQ ID NO: 18, 24, and 29 for the variable light chain domain.
  • an antibody or antigen-binding fragment comprising the CDRs of the antibody 006-H08 are disclosed, whereby a. the variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7 and 13 and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, and 29.
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 78, 24, 90; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 75, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 82, 24, 91 ; or
  • variable heavy chain contains the CDR sequences corresponding to
  • SEQ ID NO: 1 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 82, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 86, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 87, 24, 100; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 87, 24, 92; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 89, 24, 93; or
  • variable heavy chain contains the CDR sequences corresponding to
  • SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 79, 24, 101 ; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 76, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 89, 24, 90; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 100; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 97; or m.
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 98; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 83, 24, 99; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 96; or
  • variable heavy chain contains the CDR sequences corresponding to
  • SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 94; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 88, 24, 90; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 74, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 81 , 24, 95; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 75, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 77, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 18, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to
  • SEQ ID NO: 1 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 80, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 85, 24, 29; or
  • variable heavy chain contains the CDR sequences corresponding to SEQ ID NO: 1 , 7, 13, and the variable light chain contains the CDR sequences corresponding to SEQ ID NO: 84, 24, 29.
  • human antibody 006-H08, or maturated form thereof are disclosed, whereby the antibody 006-H08 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 46, and an amino acid sequence according to SEQ ID NO: 34, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 52, and an amino acid sequence according to SEQ ID NO: 40.
  • 006-H08-12-2 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 331 , and an amino acid sequence according to SEQ ID NO: 353, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 165, and an amino acid sequence according to SEQ ID NO: 143,
  • 006-H08-13-2 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 332, ,and an amino acid sequence according to SEQ ID NO: 354, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 166, and an amino acid sequence according to SEQ ID NO: 144,
  • 006-H08-13-6-1 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 333, and an amino acid sequence according to SEQ ID NO: 355, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 167, and an amino acid sequence according to SEQ ID NO: 145,
  • 006-H08-14-6-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 334, and an amino acid sequence according to SEQ ID NO: 356, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 168, and an amino acid sequence according to SEQ ID NO: 146,
  • 006-H08-15-5 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 335, and an amino acid sequence according to SEQ ID NO: 357, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 169, and an amino acid sequence according to SEQ ID NO: 147,
  • 006-H08-19-1 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 336, and an amino acid sequence according to SEQ ID NO: 358, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 170, and an amino acid sequence according to SEQ ID NO: 148
  • 006-H08-29-1 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 337, and an amino acid sequence according to SEQ ID NO: 359, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 171 , and an amino acid sequence according to SEQ ID NO: 149,
  • 006-H08-32-2 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 338, and an amino acid sequence according to SEQ ID NO: 360, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 172, and an amino acid sequence according to SEQ ID NO: 150,
  • 006-H08-33-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 339, and an amino acid sequence according to SEQ ID NO: 361 , and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 173, and an amino acid sequence according to SEQ ID NO: 151 ,
  • 006-H08-33-16-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 340, and an amino acid sequence according to SEQ ID NO: 362, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 174, and an amino acid sequence according to SEQ ID NO: 152,
  • 006-H08-35-17-1 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 341 , and an amino acid sequence according to SEQ ID NO: 363, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 175, and an amino acid sequence according to SEQ ID NO: 153,
  • 006-H08-35-17-4 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 342, and an amino acid sequence according to SEQ ID NO: 364, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 176, and an amino acid sequence according to SEQ ID NO: 154,
  • 006-H08-35-1 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 343, and an amino acid sequence according to SEQ ID NO: 365, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 177, and an amino acid sequence according to SEQ ID NO: 155,
  • o. 006-H08-36-17-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 344, and an amino acid sequence according to SEQ ID NO: 366, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 178, and an amino acid sequence according to SEQ ID NO: 156,
  • p. 006-H08-37-19-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 345, and an amino acid sequence according to SEQ ID NO: 367, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 179, and an amino acid sequence according to SEQ ID NO: 157,
  • q. 006-H08-39-7 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 346, and an amino acid sequence according to SEQ ID NO: 368, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 180, and an amino acid sequence according to SEQ ID NO: 158,
  • r. 006-H08-48-5 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 347, and an amino acid sequence according to SEQ ID NO: 369, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 181 , and an amino acid sequence according to SEQ ID NO: 159,
  • s. 006-H08-53-27-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 348, and an amino acid sequence according to SEQ ID NO: 370, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 182, and an amino acid sequence according to SEQ ID NO: 160,
  • t. 006-H08-59-30-0 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 349, and an amino acid sequence according to SEQ ID NO: 371 , and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 183, and an amino acid sequence according to SEQ ID NO:
  • u. 006-H08-63-32-4 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 350, and an amino acid sequence according to SEQ ID NO: 372, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 184, and an amino acid sequence according to SEQ ID NO: 162,
  • v. 006-H08-65-33-2 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 351 , and an amino acid sequence according to SEQ ID NO: 373, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 185, and an amino acid sequence according to SEQ ID NO: 163,
  • w. 006-H08-68-35-2 comprises a variable heavy chain domain corresponding to a nucleic acid sequence according to SEQ ID NO: 352, and an amino acid sequence according to SEQ ID NO: 374, and a variable light chain domain with a nucleic acid sequence according to SEQ ID NO: 186, and an amino acid sequence according to SEQ ID NO: 164.
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 29, or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 78, 24, 90, or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 75, 13, 82, 24, 91 , or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 82, 24, 29, or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 86, 24, 29, or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 87, 24, 100, or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 87, 24, 92 or h.
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 89, 24, 93 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 79, 24, 101 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 76, 13, 89, 24, 90 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 100 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 97 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 98 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 83, 24, 99 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 96 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 18, 24, 94 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 88, 24, 90 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 74, 13, 81 , 24, 95 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 75, 13, 18, 24, 29 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 77, 13, 18, 24, 29 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 80, 24, 29 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 85, 24, 29 or
  • the antibody contains one, two, three, four, five or six of the CDRs corresponding to SEQ ID NO: 1 , 7, 13, 84, 24, 29.
  • the antibody 006-H08 consists of an antigen-binding region that binds specifically to or has a high affinity of for one or more regions of PRLR, whose amino acid sequence is depicted by SEQ ID NO: 70, amino acid position 1 to 210, whereby the affinity is at least 100 nM, preferably less than about 100 nM, more preferably less than about 30 nM, even more ther preferred with an affinity of less than about 10 nM or even more preferred with an affinity less than 1 nM, or even more preferred with an affinity of less than 30 pM.
  • Also object of the present invention is the afore mentioned antibody 006-H08, wherein the heavy constant is a modified or unmodified lgG1 , lgG2, lgG3 or lgG4.
  • Table 1 provides a summary of dissociation constants and dissociation rates of representative antibodies of the invention, as determined by surface plasmon resonance (Biacore) with monomeric extracellular domains of PRLR (SEQ ID NO: 70) on directly immobilized antibodies.
  • Table 1 Monovalent dissociation constants and dissociation rates of the extracellular domain of human PRLR expressed in HEK293 cells determined for anti-PRLR human lgG1 molecules by surface plasmon resonance
  • the lgG1 format was used for the cell-based affinity determination, determined by fluorescence-activated cell sorting (FACS) combined with Scatchard analysis.
  • Table 2 denotes the binding strength of representative IgG antibodies on the human breast cancer cell line T47D and rat lymphoma cell line Nb2.
  • Table 2 Cell-based binding potency of anti-PRLR antibodies as determined by FACS on the human breast cancer cell line T47D and rat lymphoma cell line Nb2
  • n-CoDeR® synthetic human antibody phage display library
  • scFv and Fab fragments were investigated in parallel.
  • the targets used for scFv or Fab selection were the soluble ECD of human PRLR (amino acid positions 1 to 210 of SEQ ID NO.
  • mouse PRLR amino acid positions 1 to 210 of SEQ ID NO: 72
  • biotinylated NHS-LC biotin, Pierce
  • non-biotinylated variant as well as the human breast cancer cell line T47D expressing PRLR.
  • a combination of various approaches in phage-display technology was used to isolate high affinity, PRLR-specific, human monoclonal antibodies, by a combination of protein and whole cell pannings and through the development of specific tools.
  • the panning tools and screening methods include the ECD of the human and mouse PRLR recombinantly expressed in fusion with an Fc domain (R&D Systems, catalogue no. 1 167-PR and 1309-PR, respectively; pos. 1 -216 of SEQ ID NO: 70 and 72, respectively, each fused to the human lgG1 Fc domain, pos.
  • Antibodies of the invention are not limited to the specific peptide sequences provided herein. Rather, the invention also embodies variants of these polypeptides. With reference to the instant disclosure and conventionally available technologies and references, the skilled worker will be able to prepare, test and utilize functional variants of the antibodies disclosed herein, while appreciating that variants having the ability to bind und to functionally block PRLR fall within the scope of the present invention.
  • a variant can include, for example, an antibody that has at least one altered complementarity determining region (CDR) (hyper-variable) and/or framework (FR) (variable) domain/position, vis-a-vis a peptide sequence disclosed herein.
  • CDR complementarity determining region
  • FR framework
  • An antibody is composed of two peptide chains, each containing one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), the latter of which is in each case made up of four FR regions (VH: HFR1 , HFR2, HFR3, HFR4; VL: LFR1 , LFR2, LFR3, LFR4) and three interspaced CDRs (VL: LCDR1 , LCDR2, LCDR3; VH: HCDR1 , HCDR2, HCDR3).
  • the antigen-binding site is formed by one or more CDRs, yet the FR regions provide the structural framework for the CDRs and, hence, play an important role in antigen binding.
  • the skilled worker routinely can generate mutated or diversified antibody sequences, which can be screened against the antigen, for new or improved properties, for example.
  • Figure 12 provides the schemes for numbering each amino acid position in the variable domains VL and VH.
  • Tables 3 (VH) and 4 (VL) delineate the CDR regions for certain antibodies of the invention and compare amino acids at a given position to each other and to a corresponding consensus or "master gene" sequence, in which the CDR regions are marked with 'X'.
  • Table 5 and 6 help to assign the SEQ ID Numbers to the antibodies, antibody fragments and PRLR variants provided in this invention.
  • variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions (FR).
  • FR framework regions
  • a peptide FR domain might be altered where there is a deviation in a residue compared to a germline sequence.
  • candidate residues that can be changed include e.g. the following ones:
  • variants may be obtained by maturation, i. e. by using one antibody as starting point for optimization by diversifying one or more amino acid residues in the antibody, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties.
  • Particularly preferred is diversification of one or more amino acid residues in LCDR3 of VL, HCDR3 of VH, LCDR1 of VL and/or HCDR2 of VH.
  • Trinucleotide mutagenesis (TRIM) technology
  • TriM trinucleotide mutagenesis
  • Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide sequence described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine;
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d).
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • certain amino acids such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices
  • valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in ⁇ -pleated sheets.
  • Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns.
  • sequence identity indicates the percentage of amino acids that are identical between the sequences.
  • sequence homology indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Preferred polypeptide sequences of the invention have a sequence identity in the CDR regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%.
  • Preferred antibodies also have a sequence homology in the CDR regions of at least 80%, more preferably 90% and most preferably 95%. However, all of these disclosed molecules block prolactin receptor mediated signaling.
  • the present invention also relates to the DNA molecules that encode an antibody of the invention. These sequences include, but are not limited to, those DNA molecules set forth in SEQ ID NOs 46 and 52, and 331 to 374.
  • DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof.
  • DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homolog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, Sambrook et al., 1989 [Sambrook, J., Fritsch, E. F. and Maniatis, T.
  • Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another.
  • stringency refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, therefore, directly correlates with the structural relationships of two nucleic acid sequences. The following relationships are useful in correlating hybridization and relatedness (where T m is the melting temperature of a nucleic acid duplex):
  • T m 69.3 + 0.41 (G+C)% b.
  • the T m of a duplex DNA decreases by 1 °C with every increase of
  • ⁇ 1 and ⁇ 2 are the ionic strengths of two solutions.
  • Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the "binding" phase and the “washing” phase.
  • the probe is bound to the target under conditions favoring hybridization.
  • Stringency is usually controlled at this stage by altering the temperature.
  • the temperature is usually between 65°C and 70°C, unless short ( ⁇ 20 nt) oligonucleotide probes are used.
  • a representative hybridization solution comprises 6x SSC, 0.5% SDS, 5x Denhardt's solution and 100 ⁇ g of nonspecific carrier DNA [see Ausubel et al., section 2.9, supplement 27 (1994)]. Of course, many different, yet functionally equivalent, buffer conditions are known. Where the degree of relatedness is lower, a lower temperature may be chosen.
  • Low stringency binding temperatures are between about 25°C and 40°C.
  • Medium stringency is between at least about 40°C to less than about 65°C.
  • High stringency is at least about 65°C.
  • washing solutions typically contain lower salt concentrations.
  • One exemplary medium stringency solution contains 2X SSC and 0.1 % SDS.
  • a high stringency wash solution contains the equivalent (in ionic strength) of less than about 0.2X SSC, with a preferred stringent solution containing about 0.1 X SSC.
  • the temperatures associated with various stringencies are the same as discussed above for "binding.”
  • the washing solution also typically is replaced a number of times during washing. For example, typical high stringency washing conditions comprise washing twice for 30 minutes at 55° C and three times for 15 minutes at 60° C.
  • subject of the present invention is an isolated nucleic acid sequence that encodes the antibody and antigen-binding fragments of the present invention.
  • Another embodiment of the present invention is the afore mentioned isolated nucleic acid sequence, which encodes the antibodies of the present invention, whereby the nucleic acid sequences are as given in table 5.
  • the present invention includes nucleic acid molecules that hybridize to the molecules of set forth in Table 5 under high stringency binding and washing conditions, where such nucleic molecules encode an antibody or functional fragment thereof having properties as described herein
  • Preferred molecules are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) sequence identity with one of the DNA molecules described herein.
  • the molecules block prolactin receptor mediated signaling.
  • variants within the scope of the invention may be described with reference to the product they encode. These functionally equivalent genes are characterized by the fact that they encode the same peptide sequences found in SEQ ID No: 34-45 due to the degeneracy of the genetic code. It is recognized that variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing oligonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et al., section 2.1 1 , Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et al., J.
  • Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.
  • a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997).
  • a target DNA is cloned into a single-stranded DNA bacteriophage vehicle.
  • Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s).
  • the complementary strand is synthesized and the double stranded phage is introduced into a host.
  • Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing.
  • various methods are available that increase the probability that the progeny phage will be the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants.
  • the present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention.
  • the recombinant constructs of the present invention are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody of the invention is inserted.
  • the encoded gene may be produced by techniques described in Sambrook et al., 1989, and Ausubel et al., 1989.
  • the DNA sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in OLIGONUCLEOTIDE SYNTHESIS (1984, Gait, ed., IRL Press, Oxford), which is incorporated by reference herein in its entirety.
  • the expert in the field is able to fuse DNA encoding the variable domains with gene fragments encoding constant regions of various human IgG isotypes or derivatives thereof, either mutated or non-mutated.
  • Recombinant constructs of the invention are comprised with expression vectors that are capable of expressing the RNA and/or protein products of the encoded DNA(s).
  • the vector may further comprise regulatory sequences, including a promoter operably linked to the open reading frame (ORF).
  • the vector may further comprise a selectable marker sequence. Specific initiation and bacterial secretory signals also may be required for efficient translation of inserted target gene coding sequences.
  • the present invention further provides host cells containing at least one of the DNAs of the present invention.
  • the host cell can be virtually any cell for which expression vectors are available. It may be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, electroporation or phage infection.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • appropriate means e.g., temperature shift or chemical induction
  • a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • an object of the present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies of the present invention.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S. 4,399,216, 4,634,665 and U.S. 5,179,017, by Axel et al.).
  • Suitable selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • drugs such as G418, hygromycin or methotrexate
  • the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate
  • the neo gene confers resistance to G418.
  • Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, calcium-phosphate precipitation, and DEAE- dextran transfection.
  • Suitable mammalian host cells for expressing the antibodies, antigen binding portions, or derivatives thereof provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621]], NSO myeloma cells, COS cells and SP2 cells.
  • the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown.
  • the antibodies, antigen binding portions, or derivatives thereof can be recovered from the culture medium using standard protein purification methods.
  • Antibodies of the invention or an antigen-binding fragment thereof can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification.
  • HPLC high performance liquid chromatography
  • Antibodies of the present invention or antigen-binding fragment thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • an object of the present invention are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
  • Another object of the present invention is a method of using the host cell to produce an antibody and antigen-binding fragments, comprising culturing the host cell under suitable conditions and recovering said antibody.
  • Another object of the present invention is the antibody as described in the present invention produced with the host cells of the present invention and purified to at least 95% homogeneity by weight.
  • Endometriosis is a benign, estrogen-dependent, gynecological disorder that is characterized by the presence of endometrial tissue (glands and stroma) outside the uterine cavity. Endometriotic lesions are mainly found on the pelvic peritoneum, in the ovaries and the rectovaginal septum (Obstet. Gynecol. Clin. North. Am. 24:235-238, 1997). Endometriosis is often associated with infertility and pain symptoms such as dysmenorrhoea. In addition, many patients suffer from autoimmune diseases (Hum. Reprod. 17(19):2715-2724, 2002).
  • Adenomyosis uteri also known as endometriosis interna describes a subform of endometriosis which is restricted to the uterus.
  • endometrial glands invade the myometrium and the uterine wall.
  • endometrial fragments are flushed by retrograde menstruation into the peritoneal cavity in both, patients and healthy women (Obstet. Gynecol. 64:151-154, 1984).
  • GnRH Gonadotropin-releasing hormone
  • Aromatase inhibitors inhibit the local production of estradiol by endometriotic implants, induce apoptosis and inhibit proliferation of ectopic endometriotic fragments.
  • Selective estrogen receptor modulators have estrogen receptor antagonistic activity in normal endometrial and ectopic implants and thus lead to atrophy of implanted ectopic endometriotic tissue.
  • Progesterone receptor agonists inhibit proliferation of normal and ectopic endometrial cells, induce differentiation and apoptosis.
  • Combined oral contraceptives maintain the status quo, prevent progression of the disease, and induce atrophy of the ectopic and eutopic endometrium.
  • GnRH analogues, SERMs, and aromatase inhibitors have severe side effects and lead to hot flushes and bone loss in young women suffering from endometriosis.
  • Treatment with progesterone receptor agonists leads to ovulation inhibition, irregular menstrual bleeding followed by amenorrhoea, body weight gain and depression. Due to increased risk for venous thrombembolism, combined oral contraceptives are not indicated in women older than 35 years, smokers and individuals suffering from overweight. Surgical excision of lesions is prone to high recurrence rates.
  • the antibodies of the present invention interfere with PRLR-mediated signaling stimulated by pituitary- and locally-produced prolactin or due to activating PRLR mutations and are therefore more effective than dopamine-2- receptor agonists which interfere only with pituitary prolactin secretion.
  • an object of the present invention is the antibody 006-H08 or antigen-binding fragments as a medicament.
  • PRL and the PRLR are expressed in the uterus and play a role in normal uterine physiology; PRL can act as a potent mitogen and has an immunomodulatory role.
  • PRL/PRLR system plays a role in human endometriosis.
  • An analysis of the expression of PRL and the PRLR in endometrium of healthy women and in endometrium and lesions of patients (see Example 2) by quantitative Taqman PCR is shown in Figures 1 and 2.
  • the PRLR antibodies were successfully tested in an animal model for endometriosis interna, i.e. adenomyosis uteri in mice (see Example 20).
  • Adenomyosis is characterized by infiltrative growth of endometrial glands in the myometrial layer of the endometrium. It resembles an endometriosis form restricted to the uterus - the only form of endometriosis non-menstruating species can develop.
  • Danazol which is effective in the clinical treatment of patients suffering from endometriosis is also effective in the treatment of adenomyosis uteri (Life Sciences 51 :1 1 19-1 125, 1992).
  • danazol is an androgenic progestin and leads to severe androgenic side-effects in young women, which limits its use.
  • the antibodies of the present invention solve the problem for providing new treatments or prevention for endometriosis and exhibit lesser side effects than current standard therapies.
  • a further aspect of the present invention is to employ neutralizing PRLR antibodies and antigen-binding fragments for the treatment or prevention of endometriosis and adenomyosis (endometriosis interna).
  • Another aspect of the present invention is the use of the antibody and antigen-binding fragments as described in the present invention for the treatment or prevention of endometriosis and adenomyosis (endometriosis interna).
  • the progestogenic component mediates the contraceptive effect via negative feedback on the hypothalamic-pituitary-gonadal axis.
  • the estrogenic component guarantees a good bleeding control and potentiates the gestagenic action via induction of the progesterone receptor in target cells.
  • the locally released progestin renders the endometrium in an implantation- resistant state.
  • the cervical mucos becomes almost impermeable for sperm cells
  • Progestin only pills and implants.
  • the progestin inhibits ovulation via negative feedback on the hypothalamic- pituitary-gonadal axis.
  • the permeability of the cervical mucos for sperm cells is reduced,
  • VTE venous thromboembolism
  • Intrauterine devices and implants containing progestins only can lead to dysfunctional uterine bleeding.
  • Progestin only pills can cause irregular bleeding patterns, spotting, amenorrhea.
  • Weight gain and reductions in bone mass density are further side effects.
  • Vaginal rings can lead to vaginitis, leukorrhea or expulsion.
  • PRLR-deficient mice have been generated a few years ago (Genes Dev 1 1 :167-178, 1997). Interestingly, PRLR-deficient females, but not male mice, are completely sterile. PRLR " ' " females exhibited an arrest of egg development immediately after fertilization, i.e. they showed an arrest of preimplantation development. Only very few oocytes reached the blastocyst stage and were unable to implant in mutant females but developed to normal embryos in wildtype foster mothers after transplantation. The infertility phenotype of PRLR-deficient mice could be rescued until midterm pregnancy by progesterone supplementation.
  • PRLR-mediated signaling plays an important role in the maintenance and function of the corpus luteum producing progesterone that is necessary to allow and maintain pregnancy.
  • PRLR- deficient females, but not males exhibited a reduction in body weight associated with a reduction in abdominal fat mass and leptin levels.
  • prolactin can prevent apoptosis of cultured human granulosa cells and thus maintains early corpus luteum function as it has been demonstrated in PRLR-deficient mice (Human Reprod. 18:2672-2677,2003).
  • mice were injected with specific and unspecific PRLR antibodies and mated with males as described in example 1 1. Readouts were litter number per treatment group and litter size per animal. The experiment presented in figure 1 1 demonstrates that the treatment with the neutralizing antibody of the present invention completely prevented pregnancy in mice when tested at 30 mg/kg body weight. Compared to the afore mentioned standard approaches, female contraception with neutralizing PRLR antibodies has several advantages:
  • the antibodies can be used in smoking, overweight, and older women as well as in women suffering from lupus erythematodes (PRLR antibodies might even be beneficial for the treatment of lupus and the reduction of abdominal fat, i.e. PRLR-deficient mice had less abdominal fat).
  • Another object of the present invention is the use of PRLR-neutralizing PRLR antibodies and antigen-binding fragments for female contraception with reduced side effects compared to standard treatments.
  • Another aspect of the present invention is the use of the antibody and antigen-binding fragments as described in the present invention for female contraception with reduced side effects compared to standard treatments.
  • Benign breast disease encompasses a variety of symptoms, such as fibrocystic breast disease, fibroadenoma, mastalgia, and macrocysts. 30 - 50% of premenopausal women suffer from fibrocystic breast disease (Epidemiol Rev 19:310-327, 1997). Depending on the women's age, benign breast disease can present with distinct phenotypes (J Mammary Gland Biol Neoplasia 10:325-335, 2005): during the early reproductive phases (15- 25 years) when lobular development in the normal breast takes place, benign breast disease results in fibroadenomas. Single giant fibroadenomas as well as multiple adenomas are observed.
  • fibroadenomas are composed of stromal as well as epithelial cells and arise from lobules.
  • the breast is subject to cyclical changes during each menstrual cycle.
  • Diseased women present with cyclical mastalgia and several nodules in their breast.
  • the normal breast involutes whereas in the diseased breast macrocysts and epithelial hyperplasia with and without atypia can be observed.
  • Those forms of benign breast disease that are accompanied by enhanced epithelial cell proliferation have a higher risk for developing mammary carcinomas.
  • This risk can be up to 1 1 % if cellular atypias are present in the proliferating cell fraction (Zentralbl Gynakol 1 19:54-58, 1997). 25% of women aged 60 - 80 years also suffer from benign breast disease, often estrogen replacement therapy or adiposity are the reasons for persisting benign breast disease after menopause (Am J Obstet Gynecol 154:161 -179, 1986).
  • fibrocystic breast disease The pathophysiology of fibrocystic breast disease is determined by estrogen predominance and progesterone deficiency that results in hyperproliferation of connective tissues (fibrosis) which is followed by facultative epithelial cell proliferation.
  • the risk of breast cancer is elevated in patients exhibiting enhanced epithelial cell proliferation within the fibrocystic foci.
  • Clinically fibrocystic breast disease presents with breast pain and breast tenderness.
  • 70% of the patients with fibrocystic breast disease suffer from either corpus luteum insufficiency or anovulation (Am J Obstet 154:161 -179, 1986).
  • Corpus luteum insufficiency results in reduced progesterone levels and estrogen predominance.
  • Mastalgia (breast pain) affects about 70% of women at some time in their reproductive lifespan. Breast pain may or may not be associated with other criteria of the premenstrual syndrome. It has been demonstrated that women suffering from mastalgia respond with an excess prolactin release after stimulation of the hypothalamic pituitary axis (Clin Endocrinol 23:699-704, 1985).
  • Bromocriptine as a dopamin agonist blocks only pituitary prolactin synthesis, but not local synthesis of prolactin in the mammary epithelial cells. It is therefore only effective in those forms of mastalgia and benign breast disease that rely on elevated systemic prolactin levels.
  • Major side effects of bromocriptine are:
  • Danazol is an androgenic progestin that via its antigonadotrophic activity counteracts the estrogen predominance observed in benign breast disease.
  • Major side effects are: Menstrual irregularities, depression, acne, hirsutism, voice deepening, and hot flushes as well as weight gain.
  • Tamoxifen is a selective estrogen receptor modulator with antiestrogenic activity in the breast and estrogenic activity in the uterus. Major side effects are:
  • Progestins inhibit benign breast disease via suppression of the pituitary gonadal axis, ovulation inhibition and estrogen depletion. Estrogen depletion leads to menopausal symptoms such as bone loss and hot flushes. 5) Low dose combined oral contraceptives
  • prolactin levels have been found to be increased in one third of women with benign breast disease. Since estrogens enhance pituitary prolactin secretion, the increase in serum prolactin levels has been thought to be a consequence of the predominance of estrogens in this disease. It has been reported that an activating PRLR mutation is often present in women suffering from multiple breast adenomas - resembling a subtype of fibrocystic breast disease (Paul Kelly, Breast Congress Turin, 2007 and Proc Natl Acad Sci 105: 14533-14538;2008).
  • Elevated prolactin signaling can be the consequence of:
  • ⁇ inhibit phosphorylation of STAT5, a transcription factor that is normally activated and phosphorylated after PRLR activation.
  • neutralizing PRLR antibodies block all the above mentioned readout paradigms in a dose-dependent manner.
  • Another object of the present invention is the use of neutralizing PRLR antibodies or antigen-binding fragments for treatment of benign breast disease and mastalgia in pre- and postmenopausal women.
  • Another aspect of the present invention is the use of the antibodies or antigen-binding fragments as described in the present invention for treatment of benign breast disease and mastalgia in pre- and postmenopausal women.
  • Prolactin is the main hormone involved in lactation after child birth. This is evidenced by the phenotype of PRLR-deficient mice. Even heterozygous mice have severe lactational problems and are completely unable to nurse their offspring (Frontiers in Neuroendocrinology 22:140-145, 2001 ).
  • bromocriptine is used to inhibit lactation after child birth.
  • these compounds can provoke severe side effects such as nausea, vomiting, edema, hypotension, dizziness, hair loss, headache, and halluzinations.
  • dopamine receptor agonists are not indicated in women suffering from cardiovascular disease and hypertension.
  • a further disadvantage of bromocriptine is its short half life time requiring drug intake 4-6 times daily over a period of 14 days.
  • mice were mated with males. After birth, littersize was adjusted to 8 animals, and females were treated with specific and unspecific antibodies directed against the PRLR as described in example 15. As a measure for maternal lactation capacity, weight of the offspring was monitored daily. Readouts are desribed in detail in example 15 and results are depicted in figure 14A-D. Neutralizing PRLR antibodies show a dose- dependent inhibition of lactation and lead to mammary gland involution and reduced milk protein production.
  • Another object of the present invention is the use of neutralizing PRLR antibodies for inhibition of lactation.
  • Another object of the present invention is the use of the antibody 006-H08 and antigen- binding fragments as described in the present invention for inhibition of lactation.
  • Benign prostate hyperplasia is the fourth most prevalent healthcare condition in older men. Prostate enlargement is an age-dependent progressive condition that affects more than 50% of men aged ⁇ 50 years of age. BPH is characterized by hyperplasia of prostatic stromal and epithelial cells, resulting in the formation of large discrete nodules in the periurethral region of the prostate which compresses the urethral canal. Thus, impairment of urine flow is one major consequence of BPH.
  • Standard therapies for BPH encompass:
  • a1 -adrenergic receptor antagonists e.g. tamsulosin, alfuzosin, terazosin, doxazosin
  • a1 -adrenergic receptor antagonists e.g. tamsulosin, alfuzosin, terazosin, doxazosin
  • tamsulosin, alfuzosin, terazosin, doxazosin relief the BPH symptoms in the lower urinary tract. They decrease bladder outlet obstruction by blocking alpha-receptor-mediated stimulation of prostate smooth muscle.
  • Major side-effects are vasodilatory adverse events, dizziness and ejaculation failure.
  • reductase inhibitors e.g. finasteride
  • 5a-reductase inhibitors prevent the formation of dihydrotestosterone, the active form of testosterone in the prostate, which is responsible for the enlargement of the prostate.
  • Major side-effects are sexual dysfunction, such as erectile disorders and decreased libido.
  • This surgical treatment is associated with high morbidity. Side-effects are bleeding, incontinence, stricture formation, loss of ejaculation, and bladder perforation.
  • a stent is inserted into the prostatic part of the urethra to guarantee proper urine flow.
  • Major side-effects are encrustation, urinary tract infection, and migration of the stent.
  • stents have to be removed before any transurethral manipulation.
  • PRL and the PRLR act in an autocrine/paracrine way (J. Clin. Invest. 99:618 pp, 1997) within the prostate.
  • the PRLR is highly expressed in the prostate gland (Example 3, Figure 3). Variation of PRLR protein expression was observed in rat prostate tissue after hormonal depletion and treatment (Example 4, Figure 4). In addition to the PRLR, the prostate cells express also prolactin.
  • Example 17 male Balb/c mice received pituitary isografts under the kidney capsule and developed benign prostate hyperplasia. The effect of neutralizing prolactin receptor antibodies and unspecific antibodies on benign prostate hyperplasia was tested in this model. Readout paradigms are described in Example 17. As depicted in Figure 16, neutralizing PRLR antibodies inhibit benign prostate growth and are therefore suitable for the treatment of benign prostate hyperplasia. Another object of the present invention is the use of neutralizing PRLR antibodies or antigen-binding fragments for treatment of benign prostate hyperplasia.
  • Another aspect of the present invention is the use of the antibody or antigen-binding fragments as described in the present invention for treatment of benign prostate hyperplasia.
  • Scalp hair growth in cycles the anagen phase is characterized by active hair growth, the catagen phase shows involution and is followed by the telogen phase (resting).
  • the exogen phase the release of the dead hair
  • Hair loss can be the consequence of disturbed hair growth in any phase.
  • Telogen hair loss can have many triggers (physiological and emotional stress, medical conditions, iron and zinc deficiency), importantly androgenic alopecia in its early stages shows telogen hair shedding (Cleveland clinic journal of medicine 2009;76:361 -367).
  • Anagen hair loss is often the consequence of radiation or chemotherapy.
  • Minoxidil and Finasteride are used for the treatment of androgenetic hair loss, whereas glucocorticoids are used for alopecia areata. In general, all of these treatments have side-effects (finasteride: libido loss and impotence in men, glucocorticoids: diabetes, weight gain, osteoporosis), and the problem of treating hair loss has not been completely solved.
  • Example 17 In the experiments as described in Example 17 (benign prostate hyperplasia), animals receiving pituitary isografts, were shaved. In the course of these experiments, it was unexpectedly discovered that animals which received pituitary isografts showed a severe impairment of hair regrowth in the shaved area. Treatment with neutralizing PRLR antibodies but not with unspecific antibodies stimulated hair growth (Figure 17). This observation demonstrates that elevated prolactin receptor-mediated signaling is involved in hair loss. To analyze this in more detail, further shaving experiments in close analogy to previously described experiments were performed (British Journal of dermatology 2008; 159:300-305). These additional shaving experiments are described in Example 18. The experiments demonstrate that neutralising PRLR antibodies stimulate hair growth in hyper- and normoprolactinemic male and female mice.
  • the antibodies of the present invention solve the problem for providing new treatments for hyper- and normoprolactinemic hair loss in women and men.
  • a further aspect of the present invention is to employ neutralizing PRLR antibodies or antigen-binding fragments for the treatment or prevention of hyper- and normoprolactinemic hair loss.
  • Another aspect of the present invention is the use of the antibody or antigen-binding fragments as described in the present invention for treatment or prevention of hyperprolactinemic hair loss.
  • estradiol, or conjugated equine estrogens CEE
  • progestins for example medroxyprogesterone acetate (MPA), progesterone, drospirenone, levonorgestrel
  • MPA medroxyprogesterone acetate
  • progesterone progesterone
  • drospirenone levonorgestrel
  • Progestins have to be added to inhibit estradiol-activated uterine epithelial cell proliferation.
  • addition of progestins increases mammary epithelial cell proliferation. Since both, normal as well as cancerous mammary epithelial cells respond with proliferation towards combined estrogen plus progestin treatment, the relative risk of breast cancer was found to be increased after CEE plus MPA treatment (JAMA 233:321 -333;2002).
  • Neutralizing PRLR antibodies when administered every month or every second month to women under combined hormone therapy will inhibit enhanced breast epithelial cell proliferation.
  • Example 19 a previously developed mouse model for the quantitative analysis of progestin effects in the uterus and the breast was employed (Endocrinology 149:3952-3959,2008). Mice were ovariectomized and were treated 14 days after ovariectomy for three weeks with vehicle or 100 ng estradiol plus 100 mg/kg progesterone to mimick hormone replacement therapy. Animals were treated once weekly with specific PRLR (10 mg/kg or 30 mg/kg) or unspecific antibodies (30 mg/kg). The effects of neutralizing PRLR antibodies on proliferative activity in the breast under combined hormone therapy were analyzed.
  • the antibodies of the present invention solve the problem for treating enhanced breast epithelial cell proliferation observed under combined hormone therapy.
  • Another object of the present invention is the use of neutralizing PRLR antibodies or antigen-binding fragments in combined hormone therapy (i.e. estrogen + progestin therapy) to inhibit mammary epithelial cell proliferation.
  • combined hormone therapy i.e. estrogen + progestin therapy
  • Another aspect of the present invention is the use of the antibody or antigen-binding fragments as described in the present invention in combined hormone therapy (i.e. estrogen + progestin therapy) to inhibit mammary epithelial cell proliferation.
  • combined hormone therapy i.e. estrogen + progestin therapy
  • the target antigen human "PRLR” as used herein refers to a human polypeptide having substantially the same amino acid sequence in its extracellular domain as the amino acid positions 1 to 210 of SEQ ID NO. 70 and naturally occurring allelic and/or splice variants thereof.
  • ECD of PRLR refers to the extracellular portion of PRLR represented by the afore mentioned amino acids.
  • the target human PRLR also encompasses mutated versions of the receptor, such as the activating mutation I 146L described by Paul Kelly (Proc Natl Acad Sci U S A.105(38): 14533- 14538,2008; and oral communication Turin, 2007).
  • therapeutically effective amount is meant to refer to an amount of therapeutic or prophylactic antibody that would be appropriate to elicit the desired therapeutic or prophylactic effect or response, including alleviating some or all of such symptoms of disease or reducing the predisposition to the disease, when administered in accordance with the desired treatment regimen.
  • an antibody “binds specifically to,” is “specific to/for” or “specifically recognizes” an antigen (here, PRLR) if such an antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property.
  • PRLR antigen binding
  • “specific binding” is referring to the ability of the antibody to discriminate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • a standard ELISA assay can be carried out.
  • the scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogenperoxide).
  • the reaction in certain wells is scored by the optical density, for example, at 450 nm.
  • determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
  • binding also may refer to the ability of an antibody to discriminate between the target antigen and one or more closely related antigen(s), which are used as reference points. Additionally, “specific binding” may relate to the ability of an antibody to discriminate between different parts of its target antigen, e.g. different domains, subdomains or regions of PRLR, such as epitopes in the N-terminal or in the C-terminal region of the ECD of PRLR, or between one or more key amino acid residues or stretches of amino acid residues of the ECD of PRLR.
  • the term “immunospecific” or “specifically binding” means that the antibody binds to PRLR or its ECD with an affinity K D of lower than or equal to 10 "6 M (monovalent affinity).
  • the term “high affinity” means that the K D that the antibody binds to PRLR or its ECD with an affinity K D of lower than or equal to 10 "7 M (monovalent affinity).
  • the antibody may have substantially greater affinity for the target antigen compared to other unrelated molecules.
  • the antibody may also have substantially greater affinity for the target antigen compared to homologs, e.g. at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 "3 -fold, 10 ⁇ -fold, 10 "5 -fold, 10 "6 -fold or greater relative affinity for the target antigen.
  • affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan.
  • the affinity data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51 :660 (1949).
  • antibodies antagonize prolactin mediated signaling is meant to refer to a blockade of prolactin receptor activation by the antibodies of the present invention which leads to a complete inhibition of prolactin receptor mediated signaling.
  • antibodies compete for binding is meant to refer to a competition between one antibody and a second antibody or more antibodies for binding to the prolactin receptor.
  • antibody is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind the antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), camel bodies and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity.
  • Antibodies may carry different constant domains (Fc domains) on their heavy chain preferably derived from lgG1 , lgG2, or lgG4 isotypes (see below). Mutations for modification of effector functions may be introduced.
  • aglycosylation of lgG1 may be achieved by mutating asparagine to alanine or asparagine to glutamine at amino acid position 297, which has been reported to abolish antibody-derived cell-mediated cytotoxicity (ADCC) (Sazinsky et al., Proc. Nat. Acad. Sci. 105 (51 ): 20169, 2008; Simmons et al., J.
  • ADCC antibody-derived cell-mediated cytotoxicity
  • the tendency of human lgG2 molecules to form heterogeneous covalent dimers may be circumvented by exchanging one of the cysteines at position 127, 232 and 233 to serine (Allen et al., Biochemistry, 2009, 48 (17), pp 3755-3766).
  • An alternative format with reduced effector function may be the lgG2m4 format, derived from lgG2 carrying four lgG4-specific amino acid residue changes (An et al., mAbs 1 (6), 2009).
  • Antibody fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies and are described further below.
  • Nonlimiting examples of monoclonal antibodies include murine, chimeric, humanized, human, and Human EngineeredTM immunoglobulins, antibodies, chimeric fusion proteins having sequences derived from immunoglobulins, or muteins or derivatives thereof, each described further below. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [1975, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the “monoclonal antibodies” may also be recombinant, chimeric, humanized, human, Human EngineeredTM, or antibody fragments, for example.
  • immunoglobulin or “native antibody” is a tetrameric glycoprotein.
  • each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a "variable” region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the carboxy- terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains.
  • Heavy chains are classified as mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), alpha (a), and epsilon ( ⁇ ), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Several of these may be further divided into subclasses or isotypes, e.g. lgG1 , lgG2, lgG3, lgG4, IgAI and lgA2.
  • Different isotypes have different effector functions; for example, lgG1 and lgG3 isotypes often have ADCC activity.
  • Human light chains are classified as kappa (K) and lambda ( ⁇ ) light chains.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • a “functional fragment” or "antigen-binding antibody fragment” of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen- binding region.
  • An "antigen-binding region" of an antibody typically is found in one or more hypervariable region(s) of an antibody, i.e., the CDR-1 , -2, and/or -3 regions; however, the variable "framework" regions can also play an important role in antigen- binding, such as by providing a scaffold for the CDRs.
  • the "antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 1 1 1 of VH, and particularly preferred are the complete VL and VH chains [amino acid positions 1 to 109 of VL and 1 to 1 13 of VH, while numbering of amino acid positions occurs according to the Kabat database (Johnson and Wu, Nucleic Acids Res., 2000, 28, 214-218].
  • a preferred class of immunoglobulins for use in the present invention is IgG.
  • hypervariable region refers to the amino acid residues of the variable domains VH and VL of an antibody or functional fragment which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region" or CDR [i.e., residues 24-34 (LCDR1 ), 50- 56 (LCDR2) and 88-97 (LCDR3) in the light chain variable domain and 29-36 (HCDR1 ), 48-66 (HCDR2) and 93-102 (HCDR3) in the heavy chain variable domain as described in Fig.
  • residues from a hypervariable loop i.e., residues 26-32 (within LCDR1 ), 50-52 (within LCDR2) and 91-96 (within LCDR3) in the light chain variable domain and 26-32 (within HCDR1 ), 53- 55 (within HCDR2) and 96-101 (within HCDR3) in the heavy chain variable domain as described by Chothia et al., J. Mol.Biol. 196: 901 - 917 (1987)].
  • Nonlimiting examples of antibody fragments include Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, diabodies, triabodies, tetrabodies, minibodies, linear antibodies (Zapata et al., Protein Eng.,8(10):1057-1062 (1995)); chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or muteins or derivatives thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen-binding to the polypeptide, such as a CDR sequence, as long as the antibody retains the desired biological activity; and multispecific antibodies formed from antibody fragments (C
  • Fv F(ab')2 fragment
  • An "Fv” fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen has the ability to recognize and bind antigen.
  • Single-chain Fv or “sFv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. "Framework" or FR residues are those variable domain residues other than the hypervariable region residues.
  • constant region refers to the portion of the antibody molecule that confers effector functions.
  • mutant or “variant” can be used interchangeably and refers to the polypeptide sequence of an antibody that contains at least one amino acid substitution, deletion, or insertion in the variable region or the portion equivalent to the variable region, provided that the mutein or variant retains the desired binding affinity or biological activity. Muteins may be substantially homologous or substantially identical to the parent antibody.
  • derivative refers to antibodies covalently modified by such techniques as ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of non-natural amino acids.
  • a "human” antibody or functional human antibody fragment is hereby defined as one that is not chimeric or “humanized” and not from (either in whole or in part) a non- human species.
  • a human antibody or functional antibody fragment can be derived from a human or can be a synthetic human antibody.
  • a "synthetic human antibody” is defined herein as an antibody having a sequence derived, in whole or in part, in silico from synthetic sequences that are based on the analysis of known human antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and devising a polypeptide sequence utilizing the data obtained therefrom. Another example of a human antibody or functional antibody fragment is one that is encoded by a nucleic acid isolated from a library of antibody sequences of human origin (i.e., such library being based on antibodies taken from a human natural source). Examples of human antibodies include n-CoDeR-based antibodies as described by Carlsson and Soderlind Exp. Rev. Mol. Diagn. 1 (1 ), 102-108 (2001 ), Soderlin et al. Nat. Biotech. 18, 852-856 (2000) and U.S. Patent No. 6,989,250.
  • a “humanized antibody” or functional humanized antibody fragment is defined herein as one that is (i) derived from a non-human source (e.g., a transgenic mouse which bears a heterologous immune system), which antibody is based on a human germline sequence; or (ii) CDR-grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.
  • a non-human source e.g., a transgenic mouse which bears a heterologous immune system
  • CDR-grafted wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.
  • chimeric antibody refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions.
  • An antibody of the invention may be derived from a recombinant antibody gene library.
  • the development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a recombinant means for directly making and selecting human antibodies, which also can be applied to humanized, chimeric, murine or mutein antibodies.
  • the antibodies produced by phage technology are produced as antigen binding fragments - usually Fv or Fab fragments - in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
  • the Fd fragment (VH-CH1 ) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen.
  • the Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them.
  • selection of Fab by antigen binding co-selects for the Fab encoding sequences which can be amplified subsequently.
  • a procedure termed panning Fab specific for the antigen are enriched and finally isolated.
  • Such libraries may be built on a single master framework, into which diverse in vivo-formed (i. e. human-derived) CDRs are allowed to recombine as described by Carlsson and Soderlind Exp. Rev. Mol. Diagn. 1 (1 ), 102-108 (2001 ), Soderlin et al. Nat. Biotech. 18, 852-856 (2000) and U.S. Patent No. 6,989,250.
  • an antibody library may be based on amino acid sequences that have been designed in silico and encoded by nucleic acids that are synthetically created.
  • an antibody of this invention may come from animals.
  • Such an antibody may be humanized or Human Engineered summarized in WO08/022295 (Novartis); such an antibody may come from transgenic animals [see also WO08/022295 (Novartis)].
  • different 'forms' of antigen are hereby defined as different protein molecules resulting from different translational and posttranslational modifications, such as, but not limited to, differences in splicing of the primary prolactin receptor transcript, differences in glycosylation, and differences in posttranslational proteolytic cleavage.
  • the term 'epitope' includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Two antibodies are said to 'bind the same epitope' if one antibody is shown to compete with the second antibody in a competitive binding assay, by any of the methods well known to those of skill in the art, and if preferably all amino acids of the epitope are bound by the two antibodies.
  • the term 'maturated antibodies' or 'maturated antigen-binding fragments' such as maturated Fab variants includes derivatives of an antibody or antibody fragment exhibiting stronger binding - i. e. binding with increased affinity - to a given antigen such as the extracellular domain of the PRLR.
  • Maturation is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment leading to this affinity increase.
  • the maturation process is the combination of molecular biology methods for introduction of mutations into the antibody and screening for identifying the improved binders. Therapeutic Methods
  • Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody contemplated by the invention.
  • a "therapeutically effective" amount hereby is defined as the amount of an antibody that is of sufficient quantity to block proliferation of PRLR-positive cells in a treated area of a subject either as a single dose or according to a multiple dose regimen, alone or in combination with other agents, which leads to the alleviation of an adverse condition, yet which amount is toxicologically tolerable.
  • the subject may be a human or non- human animal (e.g., rabbit, rat, mouse, monkey or other lower-order primate).
  • An antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified.
  • an antibody could be conjugated to an immunotoxin or radioisotope to potentially further increase efficacy.
  • inventive antibodies can be used as a therapeutic or a diagnostic tool in a variety of situations where PRLR is undesirably highly expressed.
  • Disorders and conditions particularly suitable for treatment with an antibody of the inventions are endometriosis, adenomyosis, non-hormonal female fertility 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.
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • An antibody of the invention can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration.
  • an antibody of the invention might be administered by pulse infusion, with, e.g., declining doses of the antibody.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered.
  • the skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated. Determining a therapeutically effective amount of the novel polypeptide, according to this invention, largely will depend on particular patient characteristics, route of administration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp.
  • determining a therapeutically effective amount will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples.
  • the present invention also relates to pharmaceutical compositions which may comprise PRLR antibodies, alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • the present invention also relates to the administration of pharmaceutical compositions. Such administration is accomplished parenterally.
  • Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxilliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds.
  • compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the parenteral administration also comprises methods of parenteral delivery which also include intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, and intraventricular, intravenous, intraperitoneal, intrauterine, vaginal, or intranasal administration.
  • Kits The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the afore mentioned compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
  • kits may contain DNA sequences encoding the antibodies of the invention.
  • the DNA sequences encoding these antibodies are provided in a plasmid suitable for transfection into and expression by a host cell.
  • the plasmid may contain a promoter (often an inducible promoter) to regulate expression of the DNA in the host cell.
  • the plasmid may also contain appropriate restriction sites to facilitate the insertion of other DNA sequences into the plasmid to produce various antibodies.
  • the plasmids may also contain numerous other elements to facilitate cloning and expression of the encoded proteins. Such elements are well known to those of skill in the art and include, for example, selectable markers, initiation codons, termination codons, and the like.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a lyophilized powder in 1 mM-50 mM histidine, 0.1 %-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • compositions comprising a compound of the invention formulated in an acceptable carrier
  • they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency and method of administration.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, i.e. treatment of a particular disease state characterized by PRLR expression.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., lymphoma cells, or in animal models, usually mice, rats, rabbits, dogs, pigs or monkeys.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors that ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED 5 o/LD 5 o.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations what include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, eg, size and location of endometriotic lesions; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks, or once within a month depending on half- life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 2 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. See US. 4,657,760; US 5,206,344; or US 5,225,212. Those skilled in the art will employ different formulations for polynucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • Preferred specific activities for for a radiolabeled antibody may range from 0.1 to 10 mCi/mg of protein (Riva et al., Clin. Cancer Res. 5:3275s-3280s, 1999; Wong et al., Clin. Cancer Res. 6:3855-3863, 2000; Wagner et al., J. Nuclear Med. 43:267-272, 2002).
  • Figure 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.
  • PRL-mRNA prolactin-mRNA
  • FIG. 1 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.
  • PRLR-mRNA prolactin receptor-mRNA
  • Figure 3 Northern blot analysis of PRLR gene expression in rat tissues. Gene expression of the PRLR revealed high expression in placenta and prostate.
  • Figure 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.
  • Figure 6 Inhibition of prolactin-induced rat lymphoma cell proliferation (NB2 cells) by neutralizing PRLR antibodies and unspecific control antibodies.
  • Figure 7 Inhibition of prolactin-stimulated STAT5 phosphorylation in T47D cells by neutralizing PRLR antibodies and unspecific control antibody. The unspecific control antibody (FITC) does not inhibit STAT5 phosphorylation in T47D cells.
  • FITC unspecific control antibody
  • Figure 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).
  • Figure 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).
  • 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 lgG1 005-C04) and 0 animals (females treated with 30 mg/kg lgG1 005-C04).
  • Figure 12 Kabat Numbering of framework amino acid positions according to Johnson and Wu (Nucleic Acids Res. 2000, 28, 214-218).
  • Figure 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.
  • Figure 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 lgG2a antibody (open circles), and from mothers treated with the neutralizing antibody 005-C04 containing murine lgG2a constant domains ( lgG2a 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 lgG2a 005-C04 from postpartal day 8 onwards.
  • Figure 14B Incremental litter weight gain from day to day expressed as percentage of litter weight on postpartal day 1 .
  • Figure 14A presents the slope of the graphs shown in Figure 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 .
  • Figure 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 lgG2a 005-C04, but not with unspecific antibodies. Gene expression was normalized to the expression of TATA box binding protein (TBP).
  • TBP TATA box binding protein
  • Figure15A Formation of side branches and alveolar like structures in a hyperprolactinemic mouse model of benign breast disease.
  • Figure 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.
  • Figure 15C Extent of STAT5 phosphorylation in a hyperprolactinemic mouse model of benign breast disease. Some phospho-STAT5-positive cells are indicated by white arrows.
  • Basuitary 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).
  • Figure 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 ( Figure 18 B) and women (Figure 18A).
  • Figure 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.
  • Neutralising PRLR antibodies are therefore suitable to treat enhanced mammary epithelial cell proliferation under combined hormone therapy, i.e. estradiol plus progesterone treatment.
  • Fab-containing E. coli supernatants were tested for binding to the extracellular domain of the human PRLR via competition to the lgG1 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.
  • Seq ID N0:1 represents amino acid sequence of HCDR1 , 006-H08
  • Seq ID NO:2 represents amino acid sequence of HCDR1 , 002-H06
  • Seq ID NO:3 represents amino acid sequence of HCDR1 , 002-H08
  • Seq ID NO:4 represents amino acid sequence of HCDR1 , 006-H07
  • Seq ID NO:5 represents amino acid sequence of HCDR1 , 001 -E06
  • Seq ID NO:6 represents amino acid sequence of HCDR1 , 005-C04
  • Seq ID NO:7 represents amino acid sequence of HCDR2, 006-H08
  • Seq ID NO:8 represents amino acid sequence of HCDR2, 002-H06
  • Seq ID NO:9 represents amino acid sequence of HCDR2, 002-H08
  • Seq ID NO: 10 represen ts amino ac d sequence o HCDR2, 006-H07
  • Seq ID NO: 1 1 represen ts amino ac d sequence o HCDR2, 001-E06
  • Seq ID NO: 12 represen ts amino ac d sequence o HCDR2, 005-C04
  • Seq ID NO: 13 represen ts amino ac d sequence o HCDR3, 006-H08, 002-H06
  • Seq ID NO: 14 represen ts amino ac d sequence o HCDR3, 002-H08
  • Seq ID NO: 15 represen ts amino ac d sequence o HCDR3, 006-H07
  • Seq ID NO: 16 represen ts amino ac d sequence o HCDR3, 001-E06
  • Seq ID NO: 17 represen ts amino ac d sequence o HCDR3, 005-C04
  • Seq ID NO: 18 represen ts amino ac d sequence o LCDR1 , 006-H08
  • Seq ID NO: 19 represen ts amino ac d sequence o LCDR1 002-H06
  • Seq ID NO:20 represen ts amino ac d sequence o LCDR1 002-H08
  • Seq ID NO:21 represen ts amino ac d sequence o LCDR1 006-H07
  • Seq ID NO:22 represen ts amino ac d sequence o LCDR1 001 - E06
  • Seq ID NO:23 represen ts amino ac d sequence o LCDR1 005- C04
  • Seq ID NO:24 represen ts amino ac d sequence o LCDR2 006- H08, 002-H08 Seq ID NO:25 represen ts amino ac d sequence o LCDR2 002- H06
  • Seq ID NO:26 represen ts amino ac d sequence o LCDR2 006-H07
  • Seq ID NO:27 represen ts amino ac d sequence o LCDR2 001 - E06
  • Seq ID NO:28 represen ts amino ac d sequence o LCDR2 005- C04
  • Seq ID NO:29 represen ts amino ac d sequence o LCDR3 006- H08
  • Seq ID NO:30 represen ts amino ac d sequence o LCDR3 002- H06, 001-E06 Seq ID NO:31 represen ts amino ac d sequence o LCDR3 002-H08
  • Seq ID NO:32 represen ts amino ac d sequence o LCDR3 006-H07
  • Seq ID NO:33 represen ts amino ac d sequence o LCDR3 005-C04
  • Seq ID NO:34 represen ts amino ac d sequence o VH, 006-H08
  • Seq ID NO:35 represen ts amino ac d sequence o VH, 002-H06
  • Seq ID NO:36 represen ts amino ac d sequence o VH, 002-H08 Seq ID NO:37 represents amino acid sequence of VH, 006-H07
  • Seq ID NO:38 represents amino acid sequence of VH, 001-E06
  • Seq ID NO:39 represents amino acid sequence of VH, 005-C04
  • Seq ID NO:40 represents amino acid sequence of VL, 006-H08
  • Seq ID NO:41 represents amino acid sequence of VL, 002-H06
  • Seq ID NO:42 represents amino acid sequence of VL, 002-H08
  • Seq ID NO:43 represents amino acid sequence of VL, 006-H07
  • Seq ID NO:44 represents amino acid sequence of VL, 001 -E06
  • Seq ID NO:45 represents amino acid sequence of VL, 005-C04
  • Seq ID NO:46 represents nucleic acid sequence VH, 006-H08
  • Seq ID NO:47 represents nucleic acid sequence VH, 002-H06
  • Seq ID NO:48 represents nucleic acid sequence VH, 002-H08
  • Seq ID NO:49 represents nucleic acid sequence VH, 006-H07
  • Seq ID NO:50 represents nucleic acid sequence VH, 001-E06
  • Seq ID NO:51 represents nucleic acid sequence VH, 005-C04
  • Seq ID NO:52 represents nucleic acid sequence VL, 006-H08
  • Seq ID NO:53 represents nucleic acid sequence VL, 002-H06
  • Seq ID NO:54 represents nucleic acid sequence VL, 002-H08
  • Seq ID NO:55 represents nucleic acid sequence VL, 006-H07
  • Seq ID NO:56 represents nucleic acid sequence VL, 001 -E06
  • Seq ID NO:57 represents nucleic acid sequence VL, 005-C04
  • Seq ID NO:58 represents amino acid sequence of VH, HE06642, Novartis (WO2008/22295)
  • Seq ID NO:59 represents amino acid sequence of VH, XHA06642, Novartis (WO2008/22295)
  • Seq ID NO:60 represents amino acid sequence of VH, XHA06983, Novartis (WO2008/22295)
  • Seq ID NO:61 represents amino acid sequence of VL, HE06642
  • Seq ID NO:62 represents amino acid sequence of VL, XHA06642 Novartis (WO2008/22295)
  • Seq ID NO:63 represents amino acid sequence of VL, XHA06983 Novartis (WO2008/22295)
  • Seq ID NO:64 represents nucleic acid sequence VH, HE06642
  • Seq ID NO:65 represents nucleic acid sequence VH, XHA06642 Novartis (WO2008/22295)
  • Seq ID NO:66 represents nucleic acid sequence VH, XHA06983 Novartis (WO2008/22295)
  • Seq ID NO:67 represents nucleic acid sequence VL, HE06642
  • Seq ID NO:68 represents nucleic acid sequence VL, XHA06642, Novartis (WO2008/22295)
  • Seq ID NO:69 represents nucleic acid sequence VL, XHA06983, Novartis (WO2008/22295)
  • 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
  • Seq lD NO:71 epresents CDS human ECD_PRLR, nucleotide position 1-630
  • Seq ID NO:72 epresents murine ECD_PRLR, amino acid position 1 - 210
  • Seq ID NO:73 epresents CDS murine ECD_PRLR, nucleotide position 1-630
  • SEQ ID NO:74 represents HCDR2, maturated 006-H08 variants, amino acid sequence SEQ ID NO:75 epresents HCDR2, maturated 006-H08 variants, amino acid sequence SEQ ID NO:76 epresents HCDR2, maturated 006-H08 variants, amino acid sequence SEQ ID NO:77 epresents HCDR2, maturated 006-H08 variants, amino acid sequence SEQ ID NO:78 epresents LCDR1 ma tu rated 006- H08 variants amino ac d sequence SEQ ID NO:79 epresents LCDR1 ma tu rated 006- H08 variants amino ac d sequence SEQ ID NO:80 epresents LCDR1 ma tu rated 006- H08 variants amino ac d sequence SEQ ID NO:81 epresents LCDR1 ma tu rated 006- H08 variants amino ac d sequence SEQ ID NO:82 epresent
  • SEQ ID NO:144 represents VH, 006-H08-13-2, amino acid sequence
  • SEQ ID NO:145 represents VH, 006-H08-13-6-1 , amino acid sequence
  • SEQ ID NO:146 represents VH, 006-H08-14-6-0, amino acid sequence
  • SEQ ID NO:147 represents VH, 006-H08-15-5, amino acid sequence
  • SEQ ID NO:148 represents VH, 006-H08-19-1 , amino acid sequence
  • SEQ ID NO:149 represents VH, 006-H08-29-1 , amino acid sequence
  • SEQ ID NO:150 represents VH, 006-H08-32-2, amino acid sequence
  • SEQ ID NO:151 represents VH, 006-H08-33-0, amino acid sequence
  • SEQ ID NO:152 represents VH, 006-H08-33-16-0, amino acid sequence
  • SEQ ID NO:153 represents VH, 006-H08-35-17-1 , amino acid sequence
  • SEQ ID NO:154 represents VH, 006-H08-35- 7-4, amino acid sequence
  • SEQ ID NO:155 represents VH, 006-H08-35-1 , amino acid sequence
  • SEQ ID NO:156 represents VH, 006-H08-36-17-0, amino acid sequence
  • SEQ ID NO:157 represents VH, 006-H08-37-19-0, amino acid sequence
  • SEQ ID NO:158 represents VH, 006-H08-39-7, amino acid sequence
  • SEQ ID NO:159 represents VH, 006-H08-48-5, amino acid sequence
  • SEQ ID NO: 160 represents VH, 006-H08-53-27-0, amino acid sequence
  • SEQ ID NO:161 represents VH, 006-H08-59-30-0, amino acid sequence
  • SEQ ID NO: 162 represents VH, 006-H08-63-32-4, amino acid sequence
  • SEQ ID NO:163 represents VH, 006-H08-65-33-2, amino acid sequence
  • SEQ ID NO: 164 represents VH, 006-H08-68-35-2, amino acid sequence
  • SEQ ID NO:165 represents VL, 006-H08-12-2, amino acid sequence
  • SEQ ID NO:166 represents VL, 006-H08-13-2, amino acid sequence
  • SEQ ID NO:167 represents VL, 006-H08-13-6-1 , amino acid sequence
  • SEQ ID NO:168 represents VL, 006-H08-14-6-0, amino acid sequence
  • SEQ ID NO:169 represents VL, 006-H08-15-5, amino acid sequence
  • SEQ ID NO:170 represents VL, 006-H08-19-1 , amino acid sequence
  • SEQ ID NO:171 represents VL, 006-H08-29-1 , amino acid sequence
  • SEQ ID NO: 172 represents VL, 006-H08-32-2, amino acid sequence
  • SEQ ID NO: 173 represents VL, 006-H08-33-0, amino acid sequence
  • SEQ ID NO: 174 represents VL, 006-H08-33-16-0
  • amino acid sequence SEQ ID NO: 175 represents VL, 006-H08-35-17-1
  • amino acid sequence SEQ ID NO: 176 represents VL, 006-H08-35-17-4
  • amino acid sequence SEQ ID NO: 177 represents VL, 006-H08-35-1
  • amino acid sequence SEQ ID NO: 178 represents VL, 006-H08-36-17-0
  • amino acid sequence SEQ ID NO: 179 represents VL, 006-H08-37-19-0
  • amino acid sequence SEQ ID NO: 180 represents VL, 006-H08-39-7
  • amino acid sequence SEQ ID NO:181 represents VL, 006-H08-48-5
  • amino acid sequence SEQ ID NO: 182 represents VL, 006-H08-53-27-0
  • amino acid sequence SEQ ID NO: 183 represents VL, 006-H08-59-3
  • SEQ ID NO:370 represents VL, 006-H08-53-27-0
  • nucleic acid sequence SEQ ID NO:371 represents VL, 006-H08-59-30-0
  • nucleic acid sequence SEQ ID NO:372 represents VL, 006-H08-63-32-4
  • nucleic acid sequence SEQ ID NO:373 represents VL, 006-H08-65-33-2
  • nucleic acid sequence SEQ ID NO:374 represents VL, 006-H08-68-35-2
  • 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 WO08/022295 represents (Novartis),.
  • Alternative targets were the ECD of PRLR C-terminally linked to six histidines or to a human lgG1-Fc domain via the linker with the amino acid sequence "isoleucine-glutamate-glycine-arginine-methionine-aspartate".
  • 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 ⁇ 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-CI 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.
  • TAA Triethylamine
  • 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.
  • 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.
  • PRLR anti-Prolactin Receptor
  • Real-timeTaqman 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.
  • the results shown in Figure 3 indicate a strong expression of the prolactin receptor in the placenta, the prostate, the ovary and the adrenal gland.
  • Rats were either castrated or remained intact. Intact animals were treated daily for 14 days with vehicle (intact), DHT (3 mg/kg) court 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 Figure 4 and indicate the hormonal regulation of the prolactin receptor in the rat prostate.
  • 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.
  • the dose-response curves and IC 5 o values are depicted in Figure 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.
  • Nb2-1 1 cells prolactin-dependent rat lymphoma cell
  • Nb2-1 1 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.
  • T47D cells were grown in RPMI containing 10% FCS and 2 mM glutamine. Cells were seeded on 24-well plates at a density of 0.5 x 10 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 ⁇ of lysisbuffer.
  • 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 Figure 8. In contrast to the unspecific antibody, 006-H08 and HE06.642 inhibited luciferase activity in HEK293 cells stably transfected with the human PRLR.
  • 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 Figure 9.
  • the antibody HE06.642 closed circles
  • the unspecific control antibody is completely inactive.
  • the antibody 005-C04 is able to block murine PRLR- mediated signaling.
  • 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.
  • the dose-response curves and IC 5 o values are depicted in Figure 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.
  • mice were mated for 7 days (day 0 - day 7).
  • 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 Figure 1 1 .
  • Figure 1 1 A shows the obtained pregnancy rates. Pregnancy rates were as follows:
  • FIG. 1 B 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,
  • mice per litter in the group of mice treated with the unspecific control antibody (10 mg/kg),
  • mice • 0 mice per litter in the group of mice treated with the neutralizing PRLR antibody lgG1 005-C04 (30 mg/kg).
  • Epitope grouping experiments were performed using Biacore by monitoring simultaneous binding of pairs of anti-PRLR antibodies to ECD-PRLR (SEQ ID NO: 70). 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.
  • NHS n-hydroxysuccinamide
  • EDC N-ethyl-N'- dimethylaminopropyl carbodiimide
  • 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 5x10 6 cells/ml in 1xPBS 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 ⁇ / well). For secondary antibody and autofluorescence controls, 50 ⁇ FACS buffer was added to appropriate wells.
  • 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 .5 x 10 6 cells per well. The next day, growth medium was renewed. On the third, day cells were starved for 1 hour in serumfree RPMI.
  • 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
  • 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 Figure 14A, B). The results are depicted in Figure 14.
  • Figure 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.
  • Figure 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.
  • Figure 14B shows the slope of the graphs depicted in Figure 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.
  • Black arrows in Figure 14C point to fatty islands in the mammary gland tissue (see dose-dependent effect of the specific antibody lgG2a 005- C04 on the extent of mammary gland involution ( Figure 14C)).
  • 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 ( Figure 14D).
  • Gene expression was normalized to the expression of TATA-box binding protein (TBP).
  • TBP TATA-box binding protein
  • the neutralizing PRLR antibody lgG2a 005-C04 dose- dependently decreased milk protein expression whereas the unspecific antibody (10 mg/kg) was without any significant effect.
  • the neutralizing PRLR antibody lgG2a 005-C04 dose-dependently blocked lactation and lead to mammary gland involution in lactating mice demonstrating its usefulness for lactation inhibition.
  • Neutralizing PRLR antibodies are suitable for the treatment of benign breast disease
  • 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.
  • Experimental group size was 8-10 animals.
  • 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 ( Figure 15A).
  • 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.
  • treatment with the neutralizing antibody lgG1 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 lgG1 005-C04 at 30 mg/kg completely inhibits side branching in 9 out of 9 animals receiving a pituitary isograft.
  • FIG. 15B Histological analysis and BrdU immunostaining are depicted in Figure 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 Figure 15B.
  • Mice treated with the neutralizing antibody lgG1 005-C04 (30 mg/kg body weight) showed almost complete inhibition of epithelial cell proliferation in mammary glands.
  • Neutralizing PRLR anibodies stimulated hair growth under hyperprolactinemic conditions. Representative photographs are shown in Figure 17. Therefore neutralizing PRLR antibodies can be used for the treatment of hyperprolactinemic hair loss.
  • mice The following experimental groups were used (group size was 6 mice):
  • 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.
  • the experiment comprised the following groups:
  • Neutralising PRLR antibodies are therefore suitable to treat enhanced mammary epithelial cell proliferation under combined hormone therapy, i.e. estradiol plus progesterone treatment.
  • 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
  • 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
  • the experiment comprised the following experimental groups:
  • mice Animals without pituitary transplantation, i.e. normoprolactinemic mice
  • mice Animals with pituitary transplantation, i.e. hyperprolactinemic mice
  • 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.
  • 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.
  • N represents a 25% mix each of adenine, thymine, guanine, and cytosine nucleotides
  • K represents a 50% mix each of thymine and guanine nucleotides

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BR112012015852B1 (pt) 2020-12-29
RS55589B1 (sr) 2017-06-30
PT2510006T (pt) 2017-01-20
SI2510006T1 (sl) 2017-02-28
SG181511A1 (en) 2012-07-30
PT2510002T (pt) 2016-11-23
BR112012015852B8 (pt) 2021-05-25
EP2510004A1 (de) 2012-10-17
CA2783651A1 (en) 2011-06-16

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