U.S. patent application number 13/514994 was filed with the patent office on 2012-12-13 for neutralizing prolactin receptor antibodies and their therapeutic use.
This patent application is currently assigned to BAYER INTELLECTUAL PROPERTY GMBH. Invention is credited to Sandra Bruder, Christoph Freiberg, Simone Greven, Axel Harrenga, Christiane Otto, Mark Trautwein, Siegmund Wolf.
Application Number | 20120315276 13/514994 |
Document ID | / |
Family ID | 42091520 |
Filed Date | 2012-12-13 |
United States Patent
Application |
20120315276 |
Kind Code |
A1 |
Otto; Christiane ; et
al. |
December 13, 2012 |
NEUTRALIZING PROLACTIN RECEPTOR ANTIBODIES AND THEIR THERAPEUTIC
USE
Abstract
The present invention is directed to the neutralizing prolactin
receptor antibody 002-H08, and antigen binding fragments,
pharmaceutical compositions containing them and their use in the
treatment or prevention of benign disorders and indications
mediated by the prolactin receptor such as endometriosis,
adenomyosis, non-hormonal female contraception, benign breast
disease and mastalgia, lactation inhibition, benign prostate
hyperplasia, fibroids, hyper- and normoprolactinemic hair loss, and
cotreatment in combined hormone therapy to inhibit mammary
epithelial cell proliferation. The antibodies of the invention
block prolactin receptor-mediated signaling.
Inventors: |
Otto; Christiane; (Berlin,
DE) ; Wolf; Siegmund; (Berlin, DE) ; Freiberg;
Christoph; (Wuppertal, DE) ; Harrenga; Axel;
(Koln, DE) ; Greven; Simone; (Wuppertal, DE)
; Trautwein; Mark; (Wuppertal, DE) ; Bruder;
Sandra; (Wuppertal, DE) |
Assignee: |
BAYER INTELLECTUAL PROPERTY
GMBH
Monheim
DE
|
Family ID: |
42091520 |
Appl. No.: |
13/514994 |
Filed: |
November 18, 2010 |
PCT Filed: |
November 18, 2010 |
PCT NO: |
PCT/EP2010/067744 |
371 Date: |
August 29, 2012 |
Current U.S.
Class: |
424/139.1 ;
435/252.3; 435/254.2; 435/320.1; 435/331; 435/69.6; 530/387.9;
536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 15/14 20180101; A61P 5/00 20180101; A61P 21/00 20180101; C07K
2317/73 20130101; A61K 39/3955 20130101; C07K 16/28 20130101; A61P
15/18 20180101; A61P 5/10 20180101; A61P 15/00 20180101; A61P 35/00
20180101; A61P 13/08 20180101; C07K 2317/565 20130101; C07K 16/2869
20130101; C07K 2317/76 20130101; A61P 5/08 20180101; A61K 2039/505
20130101; A61P 43/00 20180101; A61P 5/24 20180101; A61P 15/08
20180101; A61P 5/30 20180101; A61K 45/06 20130101; A61P 17/14
20180101; C07K 16/2866 20130101; A61P 15/02 20180101; C07K 2317/56
20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 536/23.53; 435/320.1; 435/331; 435/254.2; 435/252.3;
435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/13 20060101 C12N015/13; C12N 15/63 20060101
C12N015/63; A61P 17/14 20060101 A61P017/14; C12N 1/19 20060101
C12N001/19; C12N 1/21 20060101 C12N001/21; C12P 21/02 20060101
C12P021/02; C07K 16/28 20060101 C07K016/28; C12N 5/10 20060101
C12N005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 10, 2009 |
EP |
09075546.3 |
Claims
1-2. (canceled)
3. 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, and whereby the
antibody or the antigen-binding fragment competes to the antibody
002-H08.
4. Antibody or antigen-binding fragment according to claim 3, 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: 36 for
the variable heavy chain domain and, identical to SEQ ID NO: 42 for
the variable light chain domain, or b. whereby 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: 3, 9, and 14 for the heavy chain domain,
and to SEQ ID NO: 20, 24, and 31 for the variable light chain
domain.
5. Antibody or antigen-binding fragment comprising the CDRs of the
antibody of claims 3, whereby the variable heavy chain contains the
CDR sequences corresponding to SEQ ID NO: 3, 9 and 14 and the
variable light chain contains the CDR sequences corresponding to
SEQ ID NO: 20, 24, and 31.
6. Antibody or antigen-binding fragment according to claims 3,
whereby the antibody 002-H08 comprises a variable heavy region
corresponding to a nucleic acid sequence according to SEQ ID NO:
48, and an amino acid sequence according to SEQ ID NO: 36, and a
variable light region with a nucleid acid sequence according to SEQ
ID NO: 54, and an amino acid sequence according to SEQ ID NO:
42.
7. Antibody or antigen-binding fragment according to claim 3,
whereby the antibody contains one, two, three, four, five or six of
the CDRs corresponding to SEQ ID NO: 3, 9, 14, 20, 24, and 31.
8. Antibody according to claims 3, whereby the antibody 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 and human polymorphic
variants of 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 preferred with an
affinity of less than about 10 nM, or even more preferred with an
affinity of less than 1 nM.
9. Antibody of claims 3 wherein the heavy constant is a modified or
unmodified IgG1, IgG2, IgG3 or IgG4.
10. An isolated nucleic acid sequence encoding an antibody or
antigen-binding fragment according to claim 3.
11. An isolated nucleic acid sequence according to claim 10,
whereby the nucleic acid sequences are according to table 5.
12. Expression vector comprising a nucleic acid sequence of claims
10.
13. Host cell comprising the vector of claim 12, 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.
14. A method of producing an antibody or antigen-binding fragment,
comprising culturing the host cell of claim 13 under suitable
conditions and recovering said antibody.
15-36. (canceled)
37. A method for treating or preventing endometriosis, adenomyosis
(endometriosis interna), hot flashes, benign breast disease,
mastalgia, benign prostate hyperplasia, hyper- and
normoprolactinemic hair loss comprising the step of administering
to a patient in need thereof an antibody according to claim 3.
38. A method of contraception or for preventing pregnancy
comprising the step of administering to a patient in need thereof
an antibody according to claim 3.
39. A method for inhibition of lactation comprising the step of
administering to a patient in need thereof an antibody according to
claim 3.
Description
[0001] The present invention is directed towards the prolactin
receptor antibody 002-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.
[0002] There is an unmet medical need for the treatment of various
benign diseases and indications such as endometriosis, adenomyosis,
non-hormonal female contraception, benign breast disease,
mastalgia, lactation inhibition, benign prostate hyperplasia,
fibroids, hyper- and normoprolactinemic hair loss, and prevention
of mammary epithelial cell proliferation in combined (i.e. estrogen
plus progestin) hormone therapy.
[0003] Prolactin (PRL) is a polypeptide hormone composed of 199
amino acids. PRL belongs to the growth hormone (GH), placental
lactogen (PL) family of polypeptide hormones and is synthesized in
lactotroph cells of the pituitary and in several extrapituitary
tissues such as lymphocytes, mammary epithelial cells, the
myometrium, and the prostate. Two different promoters regulate
pituitary and extrapituitary PRL synthesis (BioEssays 28:1051-1055,
2006).
[0004] PRL binds to the PRL receptor (PRLR), a single transmembrane
receptor belonging to the class 1 cytokine receptor superfamily
(Endocrine Reviews 19:225-268, 1998). The PRLR exists in three
different isoforms, the short, the long, and the intermediate form
that can be distinguished by the length of their cytoplasmic tails.
Upon ligand binding, a sequential process leads to PRLR activation.
PRL interacts via its binding site 1 with one PRLR molecule and
then attracts via its binding site 2 a second receptor molecule
leading to an active dimer of PRLRs. PRLR dimerization leads to the
predominant activation of the JAK/STAT (Janus Kinase/Signal
transducers and activators of transcription) pathway. Upon receptor
dimerization, JAKs (predominantly JAK2) associated with the
receptor, transphosphorylate and activate each other. In addition
the PRLR is also phosphorylated and can bind to SH2-domain
containing proteins such as STATs. Receptor bound STATs are
subsequently phosphorylated, dissociate from the receptor and
translocate to the nucleus where they stimulate transcription of
target genes. In addition, activation of the Ras-Raf-MAPK pathway
and activation of the cytoplasmic src kinase by PRLRs have been
described (for review Endocrine Reviews 19:225-268, 1998).
[0005] 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).
[0006] Currently, complete interference with PRLR-mediated
signaling is not possible. The only way to interfere with
PRLR-mediated signaling is the inhibition of pituitary PRL
secretion by use of bromocriptine and other dopamine receptor 2
agonists (Nature Clinical Practice Endocrinology and Metabolism
2(10): 571-581, 2006). These agents however, do not suppress
extrapituitary PRL synthesis that can compensate successfully for
the inhibition of pituitary PRL synthesis leading to almost
unimpaired PRLR-mediated signaling (Endocrine Reviews
19:225-268,1998). Therefore it is not surprising that dopamine type
2 receptor agonists were not beneficial in patients suffering from
breast cancer or autoimmune diseases such as systemic lupus or
rheumatoid arthritis (Breast Cancer Res. Treat. 14:289-29, 1989;
Lupus 7:414-419, 1998) although prolactin has been implicated in
these diseases. Local prolactin synthesis in breast cancer cells or
lymphocytes which plays a pivotal role in mammary carcinoma or
autoimmune diseases, respectively, was not blocked by dopamine
receptor agonists.
[0007] Despite the above-mentioned attempts to provide means for
treatment or prevention of benign diseases and indications such as
endometriosis, adenomyosis, non-hormonal female contraception,
benign breast disease and mastalgia, lactation inhibition, benign
prostate hyperplasia, fibroids, hyper- and normoprolactinemic hair
loss, and cotreatment in combined hormone therapy for the
prevention of mammary epithelial cell proliferation no compounds
are available yet to meet that need. It is therefore an object of
the present invention, to solve that problem by providing compounds
that are therapeutics for these benign diseases and
indications.
[0008] Now novel antibodies have been identified 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.
[0009] Blockade of PRLR activation by neutralizing PRLR antibodies
leads to a complete inhibition of PRLR-mediated signaling. In
contrast, dopamine receptor agonists can only interfere with
enhanced PRLR-mediated signaling in response to elevated pituitary
prolactin secretion, but not with enhanced PRLR-mediated signaling
due to an activating PRLR mutation or due to locally elevated
prolactin production.
[0010] Therefore the problem is solved by provision of the antibody
002-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.
[0011] Some PRLR antibodies have already been described in the
application WO2008/022295 (Novartis) and in the U.S. Pat. No.
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: 36, 42, 48, and 54). 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.
[0012] Therefore an object of the present invention is an antibody
or antigen-binding fragment, whereby said antibody antagonizes
prolactin receptor-mediated signaling.
[0013] The novel antibodies `006-H08`, `002-H06`, `006-H07`,
`001-E06`, `005-C04` are subject matter of corresponding
applications.
[0014] The in vivo characterization of these antibodies in murine
disease models is only possible with an antibody exhbiting
cross-reactivity to the murine prolactin receptor. Only antibody
005-C04, which is disclosed in a corresponding application, shows
sufficient cross-reactivity to the murine prolactin receptor and is
therefore the only antibody that can be used in murine in vivo
models. The results obtained with this antibody prove therefore as
a surrogate the general efficacy for all of the other neutralising
prolactin receptor antibodies which are disclosed in the
corresponding applications in the analysed disease models.
[0015] The antibodies were characterized in several cellular
systems to determine their species specificity and their potency as
well as efficacy in different readout paradigms addressing the
inactivation of PRLR-mediated signaling (see Examples 5-10).
Proliferation assays were performed with rat Nb2-11 cells (Example
6, FIG. 6) or Ba/F cells either stably transfected with the human
PRLR (Example 5, FIG. 5) or the murine PRLR (Example 10, FIG. 10).
Whereas Novartis antbody 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 corresponding application 006-H08 showed the highest potency
with regard to proliferation inhibition of Ba/F cells stably
transfected with the human PRLR (Example 5, FIG. 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. In
addition to cellular proliferation assays (Examples 5, 6, 10),
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).
Using these systems the inability of the Novartis antibody
XHA06.642 to efficiently block murine PRLR-mediated signaling
became evident again (Example 9). In contrast, the novel antibody
005-C04 (corresponding application) blocked luciferase reporter
gene activation by the murine PRLR (Example 9). STATS
phosphorylation in human T47D cells was used as additional readout
to analyze the inhibitory activity of the antibodies on the human
PRLR (Example 7, FIG. 7). As expected, unspecific antibodies were
inactive in all experimental paradigms analyzed.
[0016] 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
I146L and 176V variants being described in PNAS 105 (38), 14533,
2008, and J. Clin. Endocrinol. Metab. 95 (1), 271, 2010.
[0017] 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.
[0018] 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).
[0019] Therefore 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 002-H08 or
defined maturated variants thereof. The sequences of the antibodies
are depicted in table 5.
[0020] In yet another embodiment, an antibody or antigen-binding
fragment are disclosed, [0021] 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: 36 for the variable heavy chain domain
and, identical to SEQ ID NO: 42 for the variable light chain
domain, or [0022] b. whereby 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:
3, 9, and 14 for the heavy chain domain, and to SEQ ID NO: 20, 24,
and 31 for the variable light chain domain.
[0023] In one embodiment, antibodies and antigen-binding fragments
are disclosed comprising the CDRs of the afore mentioned antibody,
whereby the variable heavy chain contains the CDR sequences
corresponding to SEQ ID NO: 3, 9 and 14 and the variable light
chain contains the CDR sequences corresponding to SEQ ID NO: 20,
24, and 31.
[0024] In one embodiment, antibody 002-H08 and antigen binding
fragments are diclosed, whereby [0025] the antibody 002-H08
comprises a variable heavy region corresponding with a nucleic acid
sequence according to SEQ ID NO: 48, and an amino acid sequence
according to SEQ ID NO: 36, [0026] as well as a variable light
region with a nucleic acid sequence according to SEQ ID NO: 54, and
an amino acid sequence according to SEQ ID NO: 42.
[0027] In yet another embodiment, the disclosed antibody or
antigen-binding fragment contains one, two, three, four, five or
six of the CDRs corresponding to SEQ ID NO: 3, 9, 14, 20, 24, and
31.
[0028] In another embodiment of the present invention the antibody
002-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 then 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.
[0029] Also object of the present invention is the afore mentioned
antibody 002-H08, wherein the heavy constant is a modified or
unmodified IgG1, IgG2, IgG3 or IgG4.
[0030] 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-US-00001 TABLE 1 Monovalent dissociation constants and
dissociation rates of the extracellular domain of human PRLR
expressed in HEK293 cells determined for anti-PRLR human IgG1
molecules by surface plasmon resonance Antibody K.sub.D [M] kd
[1/s] 006-H08 0.7 .times. 10.sup.-9 2.4 .times. 10.sup.-4 002-H06
2.7 .times. 10.sup.-9 5.4 .times. 10.sup.-4 002-H08 1.8 .times.
10.sup.-9 2.0 .times. 10.sup.-4 006-H07 2.0 .times. 10.sup.-9 1.3
.times. 10.sup.-3 001-E06 15.8 .times. 10.sup.-9 4.8 .times.
10.sup.-3 005-C04 12.2 .times. 10.sup.-9 9.3 .times. 10.sup.-3
HE06.642 0.3 .times. 10.sup.-9 3.2 .times. 10.sup.-4 XHA06.642 1.2
.times. 10.sup.-9 1.3 .times. 10.sup.-4 XHA06.983 0.2 .times.
10.sup.-9 1.7 .times. 10.sup.-4
[0031] The IgG1 format was used for the cell-based affinity
determination, determined by fluorescence-activated cell sorting
(FACS) combined with Scatchard analysis.
[0032] 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-US-00002 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 EC.sub.50 [M] Antibody
T47D Nb2 006-H08 1.3 .times. 10.sup.-9 No binding 006-H07 0.4
.times. 10.sup.-9 No binding 001-E06 1.8 .times. 10.sup.-9 1.2
.times. 10.sup.-3 005-C04 1.9 .times. 10.sup.-9 0.5 .times.
10.sup.-9 HE06.642 1.8 .times. 10.sup.-9 0.9 .times. 10.sup.-9
XHA06.642 1.5 .times. 10.sup.-9 1.1 .times. 10.sup.-9 XHA06.983 0.3
.times. 10.sup.-9 No binding
Antibody Generation
[0033] To isolate a panel of antibodies able to functionally block
the human and murine PRLR, two formats of the synthetic human
antibody phage display library called n-CoDeR.RTM. from Bioinvent
(Soderlind et al. 2000, Nature BioTechnology. 18, 852-856.),
expressing scFv and Fab fragments, respectively, 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. 70) and mouse PRLR (amino acid positions 1 to 210 of SEQ ID NO:
72), applied as biotinylated (NHS-LC biotin, Pierce) and as
non-biotinylated variant as well as the human breast cancer cell
line T47D expressing PRLR.
[0034] A combination of various approaches in phage-display
technology (PDT) 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. 1167-PR and 1309-PR, respectively;
pos. 1-216 of SEQ ID NO: 70 and 72, respectively, each fused to the
human IgG1 Fc domain, pos. 100 to 330 of human IgG1), the
extracellular domain of the human PRLR recombinantly expressed in
fusion with a six-histidine tag (SEQ ID NO: 70), the HEK293 and the
murine lymphoma cell line Ba/F each stably transfected with human
and murine PRLR, respectively, and the breast cancer cell line T47D
and the rat lymphoma cell Nb2 each naturally expressing PRLR as
well as the development of panning procedures and screening assays
capable of identifying neutralizing antibodies that preferentially
bind to PRLR displayed on the cell surface and that are
cross-reactive to PRLR from mouse and rat (see example 6 and
10).
[0035] Screening was performed by first identifying binders for
human PRLR and eventually mouse PRLR in ELISA tests using
recombinantly expressed antigens. Then, cell binding of the Fab and
scFv fragments on T47D cells was examined by FACS analyses followed
by testing the neutralizing activity of these agents on
intracellular signaling. For this purpose, inhibition of
phosphorylation of PRLR, of STAT5 and of ERK1/2 in T47D cells was
determined (see example 14). The best function blocking scFvs and
Fabs were converted into full IgG1 molecules and tested for
monovalent affinities to the ECD of PRLR and for inhibitory
activity in luciferase reporter gene assays as well as in
proliferation assays with cells growing in dependence of prolactin.
The combination of these specific methods allowed the isolation of
the novel antibody `002-H08` which is subject matter of the present
invention, as well as of the antibodies `006-H08`, `002-H06`,
`006-H07`, `001-E06`, `005-C04` which are subject matter of
corresponding.
Peptide Variants
[0036] 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 and 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 complementarily determining region (CDR)
(hyper-variable) and/or framework (FR) (variable) domain/position,
vis-a-vis a peptide sequence disclosed herein. To better illustrate
this concept, a brief description of antibody structure
follows.
[0037] 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. By altering one or more amino acid
residues in a CDR or FR region, 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.
[0038] FIG. 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.
TABLE-US-00003 TABLE 3 VH Sequences |-HCDR1--| | 001-E06 VH
QVELLESGGG LVQPGGSLRL SCAASGFTFS S.YWNSWVRQ APGKGLEWVS 002-H06 VH
QVELLESGGG LVQPGGSLRL SCAASGFTFA N.YGLTWVRQ APGKGLEWVA 002-H08 VH
QVELLESGGG LVQPGGSLRL SCAASGFTFS S.YGMHWVRQ APGKGLEWVS 005-C04 VH
EVQLLESGGG LVQPGGSLRL SCAASGFTFS S.YWMHWVRQ APGKGLEWVS 006-H07 VH
QVELLESGGG LVQPGGSLRL SCAASGFTFE D.HGMSWVRQ APGKGLEWVS 006-H08 VH
QVELLESGGG LVQPGGSLRL SCAASGFTFD D.YGMSWVRQ APGKGLEWVA Consensus
EVQLLESGGG LVQPGGSLRL SCAASGFTXX XXXXXXXVRQ APGKGLEWVX
----HCDR2----------| |- 001-E06 VH SVSDT.GTDT HYADSVKGRF TISRDNSKNT
LYLQMNSLRA EDTAVYYCAK 002-H06 VH VISFN.GDKK YYADSVKGRF TISRDNSKNT
LYLQMNSLRA EDTAVYYCAS 002-H08 VH GVSWN.GSRT HYADSVKGRL TISRDNSKNT
LYLQMNSLRA EDTAVYYCAR 005-C04 VH DISSA.SSYT NYADSVKGRF TISRDNSKNT
LYLQMNSLRA EDTAVYYCAR 006-H07 VH LISWDDGSNK YYADSVKGRF TISRDNSKNT
LYLQMNSLRA EDTAVYYCAT 006-H08 VH VISYD.GSNK YYADSVKGRF TISRDNSQNT
LYLQMNSLRA EDTAVYYCAS Consensus XXXXXXXXXX XXXXXXXXXF TISRDNSKNT
LYLQMNSLRA EDTAVYYCXX --HCDR3-------| 001-E06 VH TPLAYSSGWY
YFDYWGQGTL VTVSS 002-H06 VH PLES....PV AFDIWGQGTL VTVSS 002-H08 VH
G........G DFDYWGQGTL VTVSS 005-C04 VH GLDA.....R RMDYWGQGTL VTVSS
006-H07 VH SLR.....AT AFDTWGQGTL VTVSS 006-H08 VH PLES....PV
QFDIWGQGTM VTVSS Consensus XXXXXXXXXX XXXXWGQGTL VTVSS
TABLE-US-00004 TABLE 4 VL Sequences |---LCDR1-----| 001-E06 VL
DIVLTQPPSA SGTPGQRVTI SCSGSSSNIG S.NTVNWYQQ LPGTAPKLLI 002-H06 VL
QSVLTQPPSA SGTPGQRVTI SCSGSYSNIG G.NPVNWYQQ LPGTAPKLLI 002-H08 VL
QSVLTQPPSA SGTPGQRVTI SCSGSSSNIG S.NDVYWYQQ LPGTAPKLLI 005-C04 VL
QSVLTQPPSA SGTPGQRVTI SCTGSSSNIG AGYVVHWYQQ LPGTAPKLLI 006-H07 VL
QSVLTQPPSA SGTPGQRVTI SCSGSSSNIG N.NAVNWYQQ LPGTAPKLLI 006-H08 VL
DIVLTQPPSA SGTPGQRVTI SCSGSNSNIG S.NPVNWYQQ LPGTAPKLLI Consensus
QSVLTQPPSA SGTPGQRVTI SCXXXXXXXX XXXXXXWYQQ LPGTAPKLLI |LCDR2|
|----LCDR3-- 001-E06 VL YRNYQRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
QSYDSSLSG. 002-H06 VL YGNSNRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
QSYDSSLSG. 002-H08 VL YDNNKRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
QSYDSSLSGS 005-C04 VL YRNNQRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
AAWDDSLNG. 006-H07 VL YSNNQRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
AAWDDSLSG. 006-H08 VL YDNNKRPSGV PDRFSGSKSG TSASLAISGL RSEDEADYYC
QSYDTGLSG. Consensus YXXXXXXXGV PDRFSGSKSG TSASLAISGL RSEDEADYYX
XXXXXXXXXX -| 001-E06 VL SVFGGGTKLT VLGQ 002-H06 VL SVFGGGTKLT VLGQ
002-H08 VL WVFGGGTKLT VLGQ 005-C04 VL WLFGGGTKLT VLGQ 006-H07 VL
WVFGGGTKLT VLGQ 006-H08 VL WVFGGGTKLT VLGQ Consensus XXFGGGTKLT
VLGQ
TABLE-US-00005 TABLE 5 Sequences of the antibodies VH VL VH VL
HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Protein Protein Nucleotide
Nucleotide Antibody SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID 006-H08 1 7 13 18 24 29 34 40 46 52 002-H06
2 8 13 19 25 30 35 41 47 53 002-H08 3 9 14 20 24 31 36 42 48 54
006-H07 4 10 15 21 26 32 37 43 49 55 001-E06 5 11 16 22 27 30 38 44
50 56 005-C04 6 12 17 23 28 33 39 45 51 57 HE06.642 -- -- -- -- --
-- 58 61 64 67 XHA06.642 -- -- -- -- -- -- 59 62 65 68 XHA06.983 --
-- -- -- -- -- 60 63 66 69
TABLE-US-00006 TABLE 6 Sequences of PRLR variants Antibody SEQ ID
Human ECD PRLR (Protein) 70 Human ECD PRLR (Nucleotide) 71 Murine
ECD PRLR (Protein) 72 Murine ECD PRLR (Nucleotide) 73
[0039] The skilled worker can use the data in Tables 3, 4 and 5 to
design peptide variants that are within the scope of the present
invention. It is preferred that 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). For
example, a peptide FR domain might be altered where there is a
deviation in a residue compared to a germline sequence.
[0040] With reference to a comparison of the novel antibodies to
the corresponding consensus or "master gene" sequence, which are
listed in FIG. 12, candidate residues that can be changed include
e.g. the following ones: [0041] residue lysine (K) at position 75
to glutamine (Q) in VH 006-H08 (SEQ ID 34) [0042] residue leucine
(L) at position 108 to methionine (M) in VH 006-H08 (SEQ ID 34)
[0043] residue threonine (T) at position 110 to isoleucine (I) in
VH 006-H08 (SEQ ID 34) [0044] residue phenylalanine (F) at position
67 to leucine (L) in VH 002-H08 (SEQ ID 36).
[0045] Furthermore, 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. Diversification can be
done by synthesizing a collection of DNA molecules using
trinucleotide mutagenesis (TRIM) technology [Virnekas, B., Ge, L.,
Pluckthun, A., Schneider, K. C., Wellnhofer, G., and Moroney S. E.
(1994) Trinucleotide phosphoramidites: ideal reagents for the
synthesis of mixed oligonucleotides for random mutagenesis. Nucl.
Acids Res. 22, 5600].
Conservative Amino Acid Variants
[0046] 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.
[0047] For example, (a) nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; (b) polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; (c) positively charged (basic) amino acids include
arginine, lysine, and histidine; and (d) negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
Substitutions typically may be made within groups (a)-(d). In
addition, glycine and proline may be substituted for one another
based on their ability to disrupt .alpha.-helices. Similarly,
certain amino acids, such as alanine, cysteine, leucine,
methionine, glutamic acid, glutamine, histidine and lysine are more
commonly found in .alpha.-helices, while valine, isoleucine,
phenylalanine, tyrosine, tryptophan and threonine are more commonly
found in .beta.-pleated sheets. Glycine, serine, aspartic acid,
asparagine, and proline are commonly found in turns. Some preferred
substitutions may be made among the following groups: (i) S and T;
(ii) P and G; and (iii) A, V, L and I. Given the known genetic
code, and recombinant and synthetic DNA techniques, the skilled
scientist readily can construct DNAs encoding the conservative
amino acid variants.
[0048] As used herein, "sequence identity" between two polypeptide
sequences, 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%.
DNA Molecules of the Invention
[0049] 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 48,
54.
[0050] 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. (1989) Molecular Cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, USA)] and Ausubel et al., 1995 [Ausubel, F. M., Brent, R.,
Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., &
Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New
York: John Wiley and Sons].
[0051] 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. As
used herein, the term "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.sub.m is the melting temperature of a nucleic
acid duplex): [0052] a. T.sub.m=69.3+0.41(G+C)% [0053] b. The
T.sub.m of a duplex DNA decreases by 1.degree. C. with every
increase of 1% in the number of mismatched base pairs. [0054] c.
(T.sub.m).sub..mu.2-(T.sub.m).sub..mu.1=18.5 log.sub.10.mu.2/.mu.1
where .mu.1 and .mu.2 are the ionic strengths of two solutions.
[0055] 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.
[0056] First, in the binding phase, the probe is bound to the
target under conditions favoring hybridization. Stringency is
usually controlled at this stage by altering the temperature. For
high stringency, the temperature is usually between 65.degree. C.
and 70.degree. C., unless short (<20 nt) oligonucleotide probes
are used. A representative hybridization solution comprises
6.times.SSC, 0.5% SDS, 5.times. Denhardt's solution and 100 .mu.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.degree. C. and
40.degree. C. Medium stringency is between at least about
40.degree. C. to less than about 65.degree. C. High stringency is
at least about 65.degree. C.
[0057] Second, the excess probe is removed by washing. It is at
this phase that more stringent conditions usually are applied.
Hence, it is this "washing" stage that is most important in
determining relatedness via hybridization. Washing solutions
typically contain lower salt concentrations. One exemplary medium
stringency solution contains 2.times.SSC and 0.1% SDS. A high
stringency wash solution contains the equivalent (in ionic
strength) of less than about 0.2.times.SSC, with a preferred
stringent solution containing about 0.1.times.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.degree. C. and three times for 15 minutes at
60.degree. C.
[0058] Accordingly, subject of the present invention is an isolated
nucleic acid sequence that encodes the antibody and antigen-binding
fragments of the present invention.
[0059] 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.
[0060] Accordingly, the present invention includes nucleic acid
molecules that hybridize to the molecules of set forth according to
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
(from an mRNA perspective) are those that have at least 75% or 80%
(preferably at least 85%, more preferably at least 90% and most
preferably at least 95%) homology or sequence identity with one of
the DNA molecules described herein. The molecules block prolactin
receptor mediated signaling.
Functionally Equivalent Variants
[0061] Yet another class of DNA 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.
[0062] 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 2.11, Supplement 21 (1993). Overlapping oligonucleotides
may be synthesized and assembled in a fashion first reported by
Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel
et al., supra, Section 8.2. 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.
[0063] As indicated, 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). In a typical method, 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. In addition,
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.
Recombinant DNA Constructs and Expression
[0064] 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.
[0065] The encoded gene may be produced by techniques described in
Sambrook et al., 1989, and Ausubel et al., 1989. Alternatively, 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. He is able to apply recombinant DNA technology in
order to fuse both variable domains in a single chain format using
linkers such as a fifteen-amino acid stretch containing three times
glycine-glycine-glycine-glycine-serine. 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.
[0066] 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.
Bacterial Expression
[0067] 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.
[0068] 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.
[0069] In bacterial systems, 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. Therefore an object of the
present invention is an expression vector comprising a nucleic acid
sequence encoding for the novel antibodies of the present
invention.
Mammalian Expression & Purification
[0070] 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. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al. The recombinant
expression vectors can also include origins of replication and
selectable markers (see e.g., U.S. Pat. No. 4,399,216, 4,634,665
and U.S. Pat. No. 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. For example, the dihydrofolate reductase
(DHFR) gene confers resistance to methotrexate and the neo gene
confers resistance to G418.
[0071] 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.
[0072] 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. In some
embodiments, 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.
[0073] 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. See, e.g., Colligan, Current Protocols in Immunology,
or Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely
incorporated herein by reference.
[0074] 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.
[0075] Therefore 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.
[0076] 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.
[0077] Therefore 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 and Adenomyosis (Endometriosis Interna)
[0078] 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. In case of adenomyosis uteri, endometrial
glands invade the myometrium and the uterine wall.
[0079] According to the transplantation theory, endometrial
fragments are flushed by retrograde menstruation into the
peritoneal cavity in both, patients and healthy women (Obstet.
Gynecol. 64:151-154, 1984). Four main factors seem to be critically
involved in the successful establishment of endometriotic lesions
in the pelvic cavity of patients: [0080] a) In the late secretory
phase of the menstrual cycle, endometrial cells in healthy women
become apoptotic. In patients, the extent of apoptosis in
endometrial cells is clearly reduced (Fertil. Steril.
69:1042-1047,1998). Therefore, in patients there is a higher
probability than in healthy women, that endometrial fragments that
have been flushed into the peritoneal cavity by retrograde
menstruation do not die and implant successfully. [0081] b) For
successful implantation in the peritoneum and long-term survival of
the ectopic endometrial fragments, new blood vessels have to form
(British Journal of Pharmacology, 149:133-135, 2006). [0082] c)
Many patients suffer from autoimmune disease and thus have a
compromised immune system (Hum. Reprod. 17(19): 2002, 2715-2724,
2002). This may lead to the conclusion that an intact immune
response--as it is present in healthy women--may play a role for
the prevention of the establishment of endometriotic lesions.
[0083] d) Lesions have to grow and thus depend on the presence of
mitogenic stimuli and growth factors.
[0084] For the treatment of endometriosis, the following approaches
exist currently: [0085] a) Gonadotropin-releasing hormone (GnRH)
analogues: lead to suppression of ovarian estradiol synthesis and
induce atrophy of ectopic endometriotic implants that depend
critically on the presence of estradiol for growth. [0086] b)
Aromatase inhibitors: inhibit the local production of estradiol by
endometriotic implants, induce apoptosis and inhibit proliferation
of ectopic endometriotic fragments. [0087] c) 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. [0088] d) Progesterone
receptor agonists: inhibit proliferation of normal and ectopic
endometrial cells, induce differentiation and apoptosis. [0089] e)
Combined oral contraceptives: maintain the status quo, prevent
progression of the disease, and induce atrophy of the ectopic and
eutopic endometrium. [0090] f) Surgical excision of lesions.
[0091] 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.
[0092] 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.
[0093] 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 a immunomodulatory role. In the present invention it is shown
that alterations in the PRL/PRLR system play 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
FIGS. 1 and 2.
[0094] As demonstrated in FIG. 1 (PRL expression) and FIG. 2 (PRLR
expression), both PRL and its receptor are strongly upregulated in
endometriotic lesions. This discovery generates for the first time
experimental evidence that autocrine PRL signaling may play a
fundamental role in the establishment, growth, and maintenance of
endometriotic lesions.
[0095] 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 51:1119-1125, 1992).
However danazol is an androgenic progestin and leads to severe
androgenic side-effects in young women, which limits its use.
[0096] 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.
[0097] Therefore 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).
[0098] 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).
Non-Hormonal Female Contraception
[0099] Current approaches for female contraception are the
following:
[0100] a) Combined oral contraceptives containing estrogens and
progestins. [0101] 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.
[0102] b) Intrauterine devices containing progestins only. [0103]
The locally released progestin renders the endometrium in an
implantation-resistant state. In addition, the cervical mucos
becomes almost impermeable for sperm cells.
[0104] c) Progestin only pills and implants. [0105] The progestin
inhibits ovulation via negative feedback on the
hypothalamic-pituitary-gonadal axis. In addition the permeability
of the cervical mucos for sperm cells is reduced.
[0106] d) Vaginal rings containing ethinylestradiol plus
progestins
[0107] The main side-effect of combined oral contraceptives is the
elevated risk for venous thromboembolism (VTE). Moreover,
overweight or smoking women, as well as women suffering from
autoimmune diseases such as lupus and women older than 35 years
cannot use oral combined contraceptives.
[0108] Intrauterine devices and implants containing progestins only
can lead to dysfunctional uterine bleeding.
[0109] Progestin only pills can cause irregular bleeding patterns,
spotting, amenorrhea. The risk for ectopic pregnancies increases.
Weight gain and reductions in bone mass density are further side
effects.
[0110] Vaginal rings can lead to vaginitis, leukorrhea or
expulsion.
[0111] PRLR-deficient mice have been generated a few years ago
(Genes Dev 11:167-178, 1997). Interestingly, PRLR-deficient
females, but not male mice, are completely sterile. PRLR.sup.-/-
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. Obviously,
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. In addition
PRLR-deficient females, but not males, exhibited a reduction in
body weight associated with a reduction in abdominal fat mass and
leptin levels.
[0112] So far, no inactivating human PRLR mutation is known,
therefore the precise role of PRLR-mediated signaling in human
fertility is still unknown. However, there is increasing evidence
that also in humans, a minimal prolactin level is required to allow
for successful pregnancy. Patients suffering from primary
infertility due to hyperprolactinemic corpus luteum insufficiency
were treated with bromocriptin. In some cases, prolactin levels
were oversuppressed and shortened luteal phases reappeared again
(Bohnet H G et al. in Lisuride and other dopamine agonists edited
by D. B. Caine et al, Raven Press, New York, 1983). From these data
it was concluded that hyper- and hypoprolactinemic states interfere
negatively with female fertility. This can be explained by the
interaction of PRL with its receptor. In case of low prolactin
levels, there is no sufficient receptor activation, whereas in case
of hyperprolactinemia, there is also no sufficient receptor
activation, since all receptors are blocked by one prolactin
molecule and cannot dimerize anymore. In other words, the dose
response for prolactin is bell-shaped and optimal receptor
activation is achieved only in a certain concentration range. There
is evidence from a second study that lack of endometrial prolactin
expression in patients leads to early implantation failure (Human
Reprod. 19:1911-1916, 2004). Moreover, it has been shown that ex
vivo, 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).
[0113] To test the contraceptive efficacy of neutralizing PRLR
antibodies, mice were injected with specific and unspecific PRLR
antibodies and mated with males as described in example 11.
Readouts were litter number per treatment group and litter size per
animal. The experiment presented in FIG. 11 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.
[0114] Compared to the afore mentioned standard approaches, female
contraception with neutralizing PRLR antibodies has several
advantages: [0115] 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). [0116] the PRLR
antibodies do not elevate the VTE (venous thrombembolic) risk
[0117] in contrast to estrogens and progestins used in combined
oral contraception, neutralization of PRLR-mediated signaling leads
to inhibition of breast epithelial proliferation and in contrast to
hormonal approaches for fertility control might even protect users
from breast cancer.
[0118] 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.
[0119] 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 and Mastalgia
[0120] 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. These fibroadenomas are composed of
stromal as well as epithelial cells and arise from lobules. In the
mature reproductive phase (25-40 years) the breast is subject to
cyclical changes during each menstrual cycle. Diseased women
present with cyclical mastalgia and several nodules in their
breast. Later (35-55 years of age), 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 11% if cellular atypias are
present in the proliferating cell fraction (Zentralbl Gynakol
119:54-58,1997). 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).
[0121] 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. As
already mentioned, 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.
[0122] 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).
[0123] Standard therapies of benign breast disease and mastalgia
are:
1) Bromocriptine
[0124] 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:
Nausea, vomiting, edema, hypotension, dizziness, hair loss,
headache, and halluzinations
2) Danazol
[0125] 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.
3) Tamoxifen
[0126] Tamoxifen is a selective estrogen receptor modulator with
antiestrogenic activity in the breast and estrogenic activity in
the uterus. Major side effects are: postmenopausal symptoms such as
bone loss and hot flushes, ovarial cysts, and endometrial
carcinoma.
4) Progestins
[0127] 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
[0128] This treatment is not indicated in women older than 35 years
of age, smoking as well as diabetic and overweight patients
[0129] In general, 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).
[0130] Benign breast disease, mastalgia and premenstrual breast
tension rely on one common pathophysiological mechanism: enhanced
prolactin signaling. Elevated prolactin signaling can be the
consequence of: [0131] systemic hyperprolactinemia (due to
pituitary adenoma) [0132] local hyperprolactinemia (due to
prolactin synthesis in proliferating mammary gland epithelial
cells). Local hyperprolactinemia does not translate into elevated
prolactin levels in the blood. [0133] constitutively active PRLR
signaling in the presence of normal prolactin levels (due to an
activating PRLR mutation).
[0134] Given that certain forms of benign breast disease can give
rise to breast cancer there is a medical need for the treatment of
this disease.
[0135] To demonstrate the efficacy of neutralizing PRLR antibodies
in a preclinical model of benign breast disease, a mouse model
based on systemic hyperprolactinemia was employed. Adult Balb/c
mice were transplanted with pituitary isografts under the kidney
capsule as described in Example 16 (In: Methods in Mammary gland
Biology and Breast Cancer Research, 101-107,2000). Systemic
hyperprolactinemia caused enhanced epithelial cell proliferation in
the mammary gland, and stimulated sidebranching and lobuloalveolar
development in comparison to untreated virgin control mice. The
most severe forms of human fibrocystic breast diseases that bear an
enhanced risk of cancerous development are characterized by
increased epithelial cell proliferation. As described in Example
16, the neutralizing PRLR antibodies were tested in this Balb/c
mouse model in comparison to unspecific antibodies with regard to
their ability to: [0136] block sidebranching and lobuloalveolar
development [0137] inhibit mammary epithelial cell proliferation
[0138] inhibit phosphorylation of STATS, a transcription factor
that is normally activated and phosphorylated after PRLR
activation.
[0139] As demonstrated in FIG. 15A-C neutralizing PRLR antibodies
block all the above mentioned readout paradigms in a dose-dependent
manner.
[0140] Another object of the present invention is the use of
neutralizing PRLR antibodies and antigen binding fragments for
treatment of benign breast disease and mastalgia in pre- and
postmenopausal women.
[0141] Another aspect of the present invention is the use of the
antibody and antigen binding fragments as described in the present
invention for treatment of benign breast disease and mastalgia in
pre- and postmenopausal women.
Lactation Inhibition
[0142] 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).
[0143] For many reasons, women have to stop breast feeding, i.e.
maternal intake of drugs potentially dangerous to the infant,
serious infections (mastitis, nephritis), profuse postpartum
hemorrhage, and severe maternal diseases such as diabetes,
carcinoma, and debility or diseases of the newborn. Currently,
dopamine receptor agonists such as bromocriptine and lisuride are
used to inhibit lactation after child birth. However, these
compounds can provoke severe side effects such as nausea, vomiting,
edema, hypotension, dizziness, hair loss, headache, and
halluzinations. In addition 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.
[0144] To test the efficacy of the neutralizing prolactin receptor
antibodies in mice, NMRI 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
described in detail in example 15 and results are depicted in FIG.
14A-D. Neutralizing PRLR antibodies show a dose-dependent
inhibition of lactation and lead to mammary gland involution and
reduced milk protein production.
[0145] Another object of the present invention is the use of
neutralizing PRLR antibodies for inhibition of lactation.
[0146] Another object of the present invention is the use of the
antibody and antigen binding fragments as described in the present
invention for inhibition of lactation.
Benign Prostate Hyperplasia
[0147] Benign prostate hyperplasia (BPH) 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 .gtoreq.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:
[0148] a) .alpha.1-adrenergic receptor antagonists (e.g.
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.
[0149] b) 5.alpha.-reductase inhibitors (e.g. finasteride) [0150]
5.alpha.-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.
[0151] c) Transurethral Resection of the Prostate (TURP) [0152]
This surgical treatment is associated with high morbidity.
Side-effects are bleeding, incontinence, stricture formation, loss
of ejaculation, and bladder perforation.
[0153] d) Prostate Stenting [0154] 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. Moreover, stents have to be removed before
any transurethral manipulation.
[0155] As described for the mammary gland, PRL and the PRLR act in
an autocrine/paracrine way (J. Clin. Invest. 99:618 pp,1997) within
the prostate.
[0156] Clinical studies indicate that hyperprolactinemia (and
agromegaly) is associated with prostatic enlargement and stromal
accumulation of inflammatory cells. Human growth hormone can bind
to the human PRLR in the presence of zinc which might explain why
acromegaly can lead to benign prostate hyperplasia. PRL serum
levels are often elevated in patients with BPH.
[0157] Transgenic animals overexpressing the PRL gene ubiquitously,
develop severe stromal prostate hyperplasia, indicating PRL as an
important pathophysiological factor for the development of prostate
hyperplasia (Endocrinology 138:4410 pp,1997). Furthermore, local
overexpression of PRL in transgenic mice under the prostate
specific probasin promoter results in stromal expansion,
accumulation of inflammatory cells and focal epithelial dysplasia
which are basic characteristics of human BPH (Endocrinology
144:2269 pp,2003).
[0158] The PRLR is highly expressed in the prostate gland (Example
3, FIG. 3). Variation of PRLR protein expression was observed in
rat prostate tissue after hormonal depletion and treatment (Example
4, FIG. 4). In addition to the PRLR, the prostate cells express
also prolactin.
[0159] As described in 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 FIG. 16, neutralizing PRLR antibodies
inhibit benign prostate growth and are therefore suitable for the
treatment of benign prostate hyperplasia.
[0160] Another object of the present invention is the use of
neutralizing PRLR antibodies and antigen binding fragments for
treatment of benign prostate hyperplasia.
[0161] Another aspect of the present invention is the use of the
antibody and antigen binding fragments as described in the present
invention for treatment of benign prostate hyperplasia.
Hyperprolactinemic Hair Loss
[0162] Treatment of hair loss is still an unmet need. 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) coincides with the end of the telogen phase. Hair loss can be
the consequence of disturbed hair growth in any phase.
[0163] 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.
[0164] 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.
[0165] In rodents, shaving experiments in adult animals were used
to analyze the effect of compounds on hair loss by using hair
regrowth in the shaved area as readout paradigm (British Journal of
dermatology 2008;159:300-305). Shaving of adult animals (hair
mostly in telogen phase) induces the anagen phase that is
characterized by hair growth.
[0166] 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 (FIG. 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.
[0167] The antibodies of the present invention solve the problem
for providing new treatments for hyper- and normoprolactinemic hair
loss in women and men.
[0168] Therefore a further aspect of the present invention is to
employ neutralizing PRLR antibodies and antigen binding fragments
for the treatment or prevention of hyper- and normoprolactinemic
hair loss.
[0169] Another aspect of the present invention is the use of the
antibody and antigen binding fragments as described in the present
invention for treatment or prevention of hyperprolactinemic hair
loss.
Combined Hormone Therapy
[0170] For the treatment of hot flushes in postmenopausal women
still having a uterus, combinations of estrogen (estradiol, or
conjugated equine estrogens=CEE) and progestins (for example
medroxyprogesterone acetate (MPA), progesterone, drospirenone,
levonorgestrel) were used. Progestins have to be added to inhibit
estradiol-activated uterine epithelial cell proliferation. However,
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).
[0171] Neutralizing PRLR antibodies when administered every month
or every second month to women under combined hormone therapy will
inhibit enhanced breast epithelial cell proliferation.
[0172] As described in 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).
[0173] The effects of neutralizing PRLR antibodies on proliferative
activity in the breast under combined hormone therapy were
analyzed.
[0174] The antibodies of the present invention solve the problem
for treating enhanced breast epithelial cell proliferation observed
under combined hormone therapy.
[0175] Another object of the present invention is the use of
neutralizing PRLR antibodies and antigen binding fragments in
combined hormone therapy (i.e. estrogen+progestin therapy) to
inhibit mammary epithelial cell proliferation.
[0176] Another aspect of the present invention is the use of the
antibody and antigen binding fragments as described in the present
invention in combined hormone therapy (i.e. estrogen+progestin
therapy) to inhibit mammary epithelial cell proliferation.
DEFINITIONS
[0177] 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" as used herein refers to the
extracellular portion of PRLR represented by the afore mentioned
amino acids. In addition the target human PRLR also encompasses
mutated versions of the receptor, such as the activating mutation
1146L described by Paul Kelly (Proc Natl Acad Sci U S
A.105(38):14533-14538,2008; and oral communication Turin,
2007).
[0178] As used herein, the phrase "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.
[0179] As used herein, 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. In its
most general form (and when no defined reference is mentioned),
"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.
For example, 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. Typical background
(=negative reaction) may be 0.1 OD; typical positive reaction may
be 1 OD. This means the difference positive/negative can be more
than 10-fold. Typically, 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. However, "specific 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.
[0180] "Affinity" or "binding affinity" K.sub.D are often
determined by measurement of the equilibrium association constant
(ka) and equilibrium dissociation constant (kd) and calculating the
quotient of kd to ka (K.sub.D=kd/ka). The term `immunospecific` or
`specifically binding` means that the antibody binds to PRLR or its
ECD with an affinity K.sub.D of lower than or equal to 10.sup.-6M
(monovalent affinity). The term "high affinity" means that the
.sub.KD that the antibody binds to PRLR or its ECD with an affinity
K.sub.D of lower than or equal to 10.sup.-7M (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.sup.-3-fold, 10.sup.-4-fold, 10.sup.-5-fold,
10.sup.-6-fold or greater relative affinity for the target antigen.
Such affinities may be readily determined using conventional
techniques, such as by equilibrium dialysis; by using the BlAcore
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).
[0181] As used herein the phrase "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.
[0182] As used herein the phrase "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.
[0183] The term "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 IgG1, IgG2, or IgG4 isotypes (see below).
Mutations for modification of effector functions may be introduced.
Amino acid residues in the Fc-domain that play a dominant role in
the interactions with the complement protein C1q and the Fc
receptors have been identified and mutations influencing effector
functions have been described (for a review see Labrijn et al.,
Current opinion in Immunology 20:479-485, 2008). Particularly,
aglycosylation of IgG1 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. of Immunological Methods 263:
133-147, 2002). Replacement of lysine by alanine at position 322
leads to reduction of ADCC and removal of complement-derived
cytotoxicity (CDC), while simultaneous replacement of the two
leucines at position 234 and 235 by alanines leads to avoidance of
ADCC and CDC [Hezareh et al., J. of Virology, 75 (24):12161-12168,
2001]. In order to apply IgG4 isotypes as bivalent therapeutics in
vivo which retain avidity, a modification such as the
serine-to-proline exchange in the `core hinge region` (Schuurman,
J. et al. Immunology 97: 693-698, 1999) may be introduced. The
tendency of human IgG2 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 IgG2m4 format, derived from IgG2
carrying four IgG4-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 Engineered.TM. 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.
[0184] 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.
[0185] 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. For example,
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. Pat. No.
4,816,567). The "monoclonal antibodies" may also be recombinant,
chimeric, humanized, human, Human Engineered.TM., or antibody
fragments, for example.
[0186] An "immunoglobulin" or "native antibody" is a tetrameric
glycoprotein. In a naturally-occurring immunoglobulin, 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 110 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 (.mu.), delta (.DELTA.), gamma (.gamma.), alpha (.alpha.),
and epsilon (.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. IgG1, IgG2, IgG3,
IgG4, IgAl and IgA2. Different isotypes have different effector
functions; for example, IgG1 and IgG3 isotypes often have ADCC
activity. Human light chains are classified as kappa (K) and lambda
(A) light chains. Within light and heavy chains, the 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. See generally, Fundamental Immunology,
Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
[0187] 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. Preferably, 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
111 of VH, and particularly preferred are the complete VL and VH
chains [amino acid positions 1 to 109 of VL and 1 to 113 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.
[0188] The term "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. 12] and/or
those 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)].
[0189] 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. A. K Borrebaeck,
editor (1995) Antibody Engineering (Breakthroughs in Molecular
Biology), Oxford University Press; R. Kontermann & S. Duebel,
editors (2001) Antibody Engineering (Springer Laboratory Manual),
Springer Verlag). An antibody other than a "bispecific" or
"bifunctional" antibody is understood to have each of its binding
sites identical. The F(ab').sub.2 or Fab may be engineered to
minimize or completely remove the intermolecular disulphide
interactions that occur between the C.sub.H1 and C.sub.L domains.
Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab')2 fragment that has two "Fv" fragments. 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. Collectively, the
six CDRs confer antigen-binding specificity to the antibody.
However, even 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.
[0190] "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.
[0191] Preferably, 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. For a review
of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies,
vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315 (1994).
[0192] 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.
[0193] "Framework" or FR residues are those variable domain
residues other than the hypervariable region residues.
[0194] The phrase "constant region" refers to the portion of the
antibody molecule that confers effector functions.
[0195] The term "mutein" 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.
[0196] Muteins may be substantially homologous or substantially
identical to the parent antibody.
[0197] The term "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.
[0198] 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. Pat. No.
6,989,250.
[0199] 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.
[0200] The phrase "chimeric antibody," as used herein, refers to an
antibody containing sequence derived from two different antibodies
(see, e.g., U.S. Pat. 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.
[0201] "Human Engineered.TM." antibodies generated by altering the
parent sequence according to the methods set forth in Studnicka et
al., U.S. Pat. No. 5,766,886 such as the antibody represented by
SEQ ID NOs 58, 61, 64, 67 and described in patent application
WO08/022295.
[0202] 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. Typically, 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.
Thus, selection of Fab by antigen binding co-selects for the Fab
encoding sequences, which can be amplified subsequently. By several
rounds of antigen binding and re-amplification, a procedure termed
panning, Fab specific for the antigen are enriched and finally
isolated.
[0203] A variety of procedures have been described for deriving
human antibodies from phage-display libraries. 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. Pat. No. 6,989,250. Alternatively, such 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. In silico design of an antibody sequence is
achieved, for example, by analyzing a database of human sequences
and devising a polypeptide sequence utilizing the data obtained
therefrom. Methods for designing and obtaining in silico-created
sequences are described, for example, in Knappik et al., J. Mol.
Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001)
254:67; and U.S. Pat. No. 6,300,064. For a review of phage display
techniques, see WO08/022295 (Novartis).
[0204] Alternatively, 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)].
[0205] As used herein, different `forms` of antigen, e.g. PRLR, 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.
[0206] As used herein, 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.
[0207] 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
[0208] 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).
[0209] An antibody of the invention might be co-administered with
known medicaments, and in some instances the antibody might itself
be modified. For example, an antibody could be conjugated to an
immunotoxin or radioisotope to potentially further increase
efficacy. The 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.
[0210] To treat any of the foregoing disorders, pharmaceutical
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. In
addition, an antibody of the invention might be administered by
pulse infusion, with, e.g., declining doses of the antibody.
Preferably, 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.
[0211] 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. 484-528 (18th ed., Alfonso R.
Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically,
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.
Pharmaceutical Compositions and Administration
[0212] The present invention also relates to pharmaceutical
compositions which may comprise PRLR antibodies, and which can be
used, 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. In one embodiment of the
present invention, the pharmaceutically acceptable carrier is
pharmaceutically inert.
[0213] 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. In addition to the active ingredients, 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.).
[0214] Pharmaceutical formulations for parenteral administration
include aqueous solutions of active compounds. For injection, the
pharmaceutical 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. Additionally,
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.
Optionally, 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.
[0215] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0216] 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
[0217] 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.
[0218] In another embodiment, the kits may contain DNA sequences
encoding the antibodies of the invention. Preferably 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.
Manufacture and Storage
[0219] The pharmaceutical 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.
[0220] 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.
[0221] After pharmaceutical compositions comprising a compound of
the invention formulated in an acceptable carrier have been
prepared, they can be placed in an appropriate container and
labeled for treatment of an indicated condition. For administration
of PRLR antibodies, such labeling would include amount, frequency
and method of administration.
Therapeutically Effective Dose
[0222] Pharmaceutical 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.
[0223] For any compound, 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.
[0224] 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.sub.50 (the dose therapeutically effective in 50% of the
population) and LD.sub.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.sub.50/LD.sub.50. 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.sub.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.
[0225] 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.
[0226] 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 U.S. Pat.
No. 4,657,760; U.S. Pat. No. 5,206,344; or U.S. Pat. No. 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 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).
[0227] The present invention is further described by the following
examples. The examples are provided solely to illustrate the
invention by reference to specific embodiments. These
exemplifications, while illustrating certain specific aspects of
the invention, do not portray the limitations or circumscribe the
scope of the disclosed invention.
[0228] All examples were carried out using standard techniques,
which are well known and routine to those of skill in the art,
except where otherwise described in detail. Routine molecular
biology techniques of the following examples can be carried out as
described in standard laboratory manuals, such as Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.
DESCRIPTION OF THE FIGURES
[0229] FIG. 1: Expression of prolactin-mRNA (PRL-mRNA) (analyzed by
real-time TaqMan PCR analysis) in human endometrium and lesions
(ectopic tissue) from healthy women and women suffering from
endometriosis.
[0230] FIG. 2: Expression of prolactin receptor-mRNA (PRLR-mRNA)
(analyzed by real-time TaqMan PCR analysis) in human endometrium
and lesions (ectopic tissue) from healthy women and women suffering
from endometriosis.
[0231] FIG. 3: Northern blot analysis of PRLR gene expression in
rat tissues. Gene expression of the PRLR revealed high expression
in placenta and prostate.
[0232] FIG. 4: Western blot analysis of PRLR expression in rat
prostates treated with different hormones. Estradiol treatment of
intact rats and castration lead to an upregulation of PRLR protein
in rat prostates whereas dihydrotestosterone treatment of intact
rats had no impact on PRLR expression in the prostate compared to
vehicle treatment of intact animals.
[0233] FIG. 5: Inhibition of prolactin-activated Ba/F (=Baf) cell
proliferation (stably expressing the human PRLR) by neutralizing
PRLRantibodies and unspecific control antibodies. The IC.sub.50
values were determined for the following antibodies in IgG1 format:
005-C04 (closed circles): 1.29 .mu.g/ml=8.6 nM; 006-H08 (open
circles): 0.15 .mu.g/ml=1 nM; HE06.642 (closed triangles): 0.34
.mu.g/ml=2.2 nM; 002-H06 (open triangles): 0.54 .mu.g/ml=3.6 nM;
002-H08 (closed squares): 0.72 .mu.g/ml=4.8 nM; unspecific control
antibody (open squares): no inhibition of cell proliferation
[0234] FIG. 6: Inhibition of prolactin-induced rat lymphoma cell
proliferation (NB2 cells) by neutralizing PRLR antibodies and
unspecific control antibodies. The following IC.sub.50 values were
determined: XHA06.642 (closed circles): 10 .mu.g/ml=67 nM;
XHA06.983 (open circles): no effect on rat lymphoma cell
proliferation; unspecific control antibody (closed triangle): no
effect at 10 .mu.g/ml.
[0235] FIG. 7: Inhibition of prolactin-stimulated STATS
phosphorylation in T47D cells by neutralizing PRLR antibodies and
unspecific control antibody.
[0236] The unspecific control antibody (FITC) does not inhibit
STAT5 phosphorylation in T47D cells. In contrast the antibodies
XHA06.642, 005-C04 (=IgG1 005-C04), and 006-H08 (=IgG1 006-H08)
inhibit in a dose-dependent manner phosphorylation of STAT5 in T47D
cells.
[0237] FIG. 8: Effects of neutralizing PRLR antibodies and
unspecific controls on prolactin-activated luciferase reporter gene
activity using HEK293 cells stably transfected with the human
prolactin receptor (hPRLR) and transiently expressing the
luciferase gene under the control of lactogenic hormone response
elements (LHREs). The IC.sub.50 values were determined for the
following antibodies in IgG1 format: 006-H08 (closed circles): 0.83
.mu.g/ml=5.5 nM; HE06.642 (open circles): 0.63 .mu.g/ml=4.2 nM;
unspecific control antibody (closed triangle): no inhibition of
luciferase activity.
[0238] FIG. 9: Effects of neutralizing PRLR antibodies and
unspecific controls on prolactin-activated luciferase reporter gene
activity using HEK293 cells stably transfected with the murine
prolactin receptor (mPRLR) and transiently expressing the
luciferase gene under the control of lactogenic hormone response
elements (LHREs). The IC.sub.50 values were determined for the
following antibodies in IgG1 format: 005-C04 (closed triangles):
0.45 .mu.g/ml=3 nM; XHA06.642 (closed circles): >>50
.mu.g/ml>>333 nM, unspecific control antibody (open circles):
no inhibition of luciferase activity.
[0239] FIG. 10: Inhibition of prolactin-activated Ba/F (=Baf) cell
proliferation (stably expressing the murine prolactin receptor) by
neutralizing prolactin receptor antibodies and unspecific control
antibodies. The IC.sub.50 values were determined for the following
antibodies in IgG1 format: unspecific FITC antibody (closed
squares): no inhibition of cell proliferation; HE06.642 (closed
circles): >>>30 .mu.g/ml>>>200 nM; 001-E06 (open
circles): 43.7 .mu.g/ml=291 nM; 001-D07 (closed triangles): 16.5
.mu.g/ml=110 nM; 005-C04 (open triangles): 0.74 .mu.g/ml=4.9
nM.
[0240] FIG. 11: Pregnancy rates and mean litter size in female mice
treated with phosphate-buffered saline (=vehicle), unspecific
control antibody (FITC IgG1) or neutralizing antibody IgG1 005-C04
(=005-C04). Pregnancy rates were 87.5% (vehicle treated females),
75% (females treated with 10 mg/kg unspecific antibody), 100%
(females treated with 10 mg/kg IgG1 005-C04), and 0% (females
treated with 30 mg/kg IgG1 005-C04). Mean litter size was 10.9
animals (vehicle treated females), 12.3 animals (females treated
with 10 mg/kg unspecific antibody), 13 animals (females treated
with 10 mg/kg IgG1 005-C04) and 0 animals (females treated with 30
mg/kg IgG1 005-C04).
[0241] FIG. 12: Kabat Numbering of framework amino acid positions
according to Johnson and Wu (Nucleic Acids Res. 2000, 28,
214-218).
[0242] FIG. 13: FACS analysis results with selected anti-PRLR
antibodies (005-C04, 001-E06, HE06642). Binding of the antibodies
was determined at a fixed concentration on HEK293 cells expressing
the human and mouse PRLR in comparison to the parental cell line
not expressing PRLR.
[0243] FIG. 14A: Litter weight gain for each postpartal day
expressed as percentage of litter weight obtained on postpartal day
1. Weight gain of litters from untreated mothers (closed circles),
from mothers treated with 10 mg/kg unspecific murine IgG2a antibody
(open circles), and from mothers treated with the neutralizing
antibody 005-C04 containing murine IgG2a constant domains (=IgG2a
005-C04) at 10 mg/kg (closed triangles) and at 30 mg/kg (open
triangles) is shown. Arrows indicate days on which antibody
injection was performed. There is a significant reduction in weight
gain from litters of mothers treated with 30 mg/kg IgG2a
005-C04from postpartal day 8 onwards.
[0244] FIG. 14B: Incremental litter weight gain from day to day
expressed as percentage of litter weight on postpartal day 1.
Results from litters of untreated mothers (closed circles), mothers
treated with 10 mg/kg unspecific murine IgG2a antibody (open
circles), mothers treated with the neutralizing antibody 005-C04
containing murine IgG2a constant domains (=IgG2a 005-C04) at 10
mg/kg (closed triangles) and at 30 mg/kg (open triangles) are
shown. Basically FIG. 14A presents the slope of the graphs shown in
FIG. 14A. Daily weight gain in litters from untreated mothers and
mothers treated with 10 mg/kg unspecific antibody oscillates around
30% of the litter weight on postpartal day 1. In contrast,
treatment of mothers with 30 mg/kg IgG2a 005-C04leads to a
significant reduction in weight gain from day 7 onwards
(*p<0.05;***p<0.005 vs. litters from mothers treated with
unspecific antibody) whereas treatment with 10 mg/kg IgG2a
005-C04leads to a significant reduction in daily weight gain from
day 11 onwards (p<0.05 vs. litters from mothers treated with
unspecific antibody). Arrows indicate days of antibody
application.
[0245] FIG. 14C: Histological sections from mammary glands of
lactating mothers. Mammary glands from untreated mothers or mothers
treated with unspecific antibody are filled with ducts producing
milk. In contrast mammary gland involution, evidenced by the
appearance of fatty islands (black arrows), is induced
dose-dependently by the neutralizing IgG2a 005-C04antibody.
[0246] FIG. 14D: Milk protein expression in mammary glands from
lactating mothers. Expression of the milk proteins beta casein
(Csn-2), whey acidic protein (WAP), and IGF-1 is reduced in a
dose-dependent manner in mothers treated with neutralizing PRLR
antibody IgG2a 005-C04, but not with unspecific antibodies. Gene
expression was normalized to the expression of TATA box binding
protein (TBP).
[0247] FIG. 15A: Formation of side branches and alveolar like
structures in a hyperprolactinemic mouse model of benign breast
disease. The neutralizing PRLR antibody IgG1 005-C04 (=005-C04)
inhibits side branching and the formation of alveolar like
structures at 10 and 30 mg/kg in mice that received a pituitary
isograft.
[0248] FIG. 15B: Extent of epithelial hyperplasia and epithelial
cell proliferation in a hyperprolactinemic mouse model of benign
breast disease. Some BrdU-positive cells are marked by white
arrows. The neutralizing PRLR antibody IgG1 005-C04 (=005-004)
blocks epithelial hyperplasia and epithelial cell proliferation in
the mammary gland.
[0249] FIG. 15C: Extent of STAT5 phosphorylation in a
hyperprolactinemic mouse model of benign breast disease. Some
phospho-STAT5-positive cells are indicated by white arrows. The
neutralizing PRLR antibody IgG1 005-C04 (=005-C04) completely
blocks STAT5 phosphorylation when applied at a dosage of 30
mg/kg.
[0250] FIG. 16: Inhibition of prostate growth by the neutralizing
PRLR antibody 005-C04 containing murine IgG2a constant domains
(=IgG2a 005-C04). Pituitary isografting stimulates prostate growth
in comparison to untreated sham-operated mice. Treatment with
neutralizing PRLR antibodies at doses of 10 mg/kg and at doses of
30 mg/kg inhibits prostate growth (***p<0.005 vs. untreated,
sham-operated mice).
[0251] FIG. 17: Neutralizing PRLR antibodies stimulate hair growth
in the presence of hyperprolactinemia. Photographs were taken three
weeks after pituitary isografting (and shaving) from male mice used
in the experiments described in Example 17 and in FIG. 16.
Hyperprolactinemia inhibits hair regrowth in the shaved areas.
Neutralizing PRLR antibodies, but not unspecific antibodies
stimulate hair regrowth under hyperprolactinemic conditions at
doses of 10 and 30 mg/kg of 005-C04 (=IgG2a 005-004).
[0252] FIG. 18: Neutralizing PRLR antibodies but not unspecific
antibodies stimulate hair regrowth in shaved areas in hyper- and
normoprolactinemic male and female mice (Example 18). Neutralizing
PRLR antibodies are therefore suitable for the treatment of hair
loss under normo- and hyperprolactinemic conditions in men (FIG. 18
B) and women (FIG. 18A).
[0253] FIG. 19: Neutralizing PRLR antibodies but not unspecific
control antibodies inhibit enhanced epithelial cell proliferation
in the mammary gland after combined hormone therapy, i.e. combined
estrogen plus progestin therapy.
[0254] The absolute number of proliferating ductal epithelial cells
within 4 cross-sections of the mammary gland was evaluated and the
medians are depicted as horizontal bars within the figure.
Epithelial cell proliferation in ovariectomized, vehicle treated
mice is rather low (median=0). Estradiol treatment leads to some
stimulation of epithelial cell proliferation (median=9), maximal
mammary epithelial cell proliferation is observed under estrogen
plus progesterone treatment (median=144). Treatment with
neutralising prolactin receptor antibody 005-C04 (median=84 after
treatment with 10 mg/kg 005-C04; median=27 after treatment with 30
mg/kg 005-C04) but not with unspecific control antibody
(median=154) leads to a dose-dependent decrease in mammary
epithelial cell proliferation almost back to estradiol-only levels.
Neutralising PRLR antibodies are therefore suitable to treat
enhanced mammary epithelial cell proliferation under combined
hormone therapy, i.e. estradiol plus progesterone treatment.
[0255] FIG. 20: Neutralizing PRLR antibodies but not unspecific
control antibodies inhibit endometriosis interna in mice. The
results are depicted as disease scores as described in Example 20.
The median disease score for each experimental group is indicated
as a horizontal bar. Normoprolactinemic mice develop endometriosis
interna to some degree (median disease score=0.25).
Hyperprolactinemia due to pituitary isografting enhances the
disease score and more animals suffer from the disease (median
disease score=2.5). Whereas treatment with 30 mg/kg unspecific
antibody once (median score =2.5) or twice (median score=2) weekly
had no influence on the disease, treatment with specific
neutralizing antibodies shows a dose-dependent decrease in the
amount of sick animals; the median disease score in all cases in
which specific antibody was used was zero. Notably, all animals
receiving either 10 or 30 mg/kg specific antibody twice weekly were
completely cured and their disease score was significantly lower
than the disease score of normoprolactinemic mice. Neutralising
PRLR antibodies are therefore suitable to treat endometriosis
interna (=adenomyosis uteri) and endometriosis externa in women.
[0256] Seq ID NO:1 represents amino acid sequence of HCDR1, 006-H08
[0257] Seq ID NO:2 represents amino acid sequence of HCDR1, 002-H06
[0258] Seq ID NO:3 represents amino acid sequence of HCDR1, 002-H08
[0259] Seq ID NO:4 represents amino acid sequence of HCDR1, 006-H07
[0260] Seq ID NO:5 represents amino acid sequence of HCDR1, 001-E06
[0261] Seq ID NO:6 represents amino acid sequence of HCDR1, 005-C04
[0262] Seq ID NO:7 represents amino acid sequence of HCDR2, 006-H08
[0263] Seq ID NO:8 represents amino acid sequence of HCDR2, 002-H06
[0264] Seq ID NO:9 represents amino acid sequence of HCDR2, 002-H08
[0265] Seq ID NO:10 represents amino acid sequence of HCDR2,
006-H07 [0266] Seq ID NO:11 represents amino acid sequence of
HCDR2, 001-E06 [0267] Seq ID NO:12 represents amino acid sequence
of HCDR2, 005-C04 [0268] Seq ID NO:13 represents amino acid
sequence of HCDR3, 006-H08, 002-H06 [0269] Seq ID NO:14 represents
amino acid sequence of HCDR3, 002-H08 [0270] Seq ID NO:15
represents amino acid sequence of HCDR3, 006-H07 [0271] Seq ID
NO:16 represents amino acid sequence of HCDR3, 001-E06 [0272] Seq
ID NO:17 represents amino acid sequence of HCDR3, 005-C04 [0273]
Seq ID NO:18 represents amino acid sequence of LCDR1, 006-H08
[0274] Seq ID NO:19 represents amino acid sequence of LCDR1,
002-H06 [0275] Seq ID NO:20 represents amino acid sequence of
LCDR1, 002-H08 [0276] Seq ID NO:21 represents amino acid sequence
of LCDR1, 006-H07 [0277] Seq ID NO:22 represents amino acid
sequence of LCDR1, 001-E06 [0278] Seq ID NO:23 represents amino
acid sequence of LCDR1, 005-C04 [0279] Seq ID NO:24 represents
amino acid sequence of LCDR2, 006-H08, 002-H08 [0280] Seq ID NO:25
represents amino acid sequence of LCDR2, 002-H06 [0281] Seq ID
NO:26 represents amino acid sequence of LCDR2, 006-H07 [0282] Seq
ID NO:27 represents amino acid sequence of LCDR2, 001-E06 [0283]
Seq ID NO:28 represents amino acid sequence of LCDR2, 005-C04
[0284] Seq ID NO:29 represents amino acid sequence of LCDR3,
006-H08 [0285] Seq ID NO:30 represents amino acid sequence of
LCDR3, 002-H06, 001-E06 [0286] Seq ID NO:31 represents amino acid
sequence of LCDR3, 002-H08 [0287] Seq ID NO:32 represents amino
acid sequence of LCDR3, 006-H07 [0288] Seq ID NO:33 represents
amino acid sequence of LCDR3, 005-C04 [0289] Seq ID NO:34
represents amino acid sequence of VH, 006-H08 [0290] Seq ID NO:35
represents amino acid sequence of VH, 002-H06 [0291] Seq ID NO:36
represents amino acid sequence of VH, 002-H08 [0292] Seq ID NO:37
represents amino acid sequence of VH, 006-H07 [0293] Seq ID NO:38
represents amino acid sequence of VH, 001-E06 [0294] Seq ID NO:39
represents amino acid sequence of VH, 005-C04 [0295] Seq ID NO:40
represents amino acid sequence of VL, 006-H08 [0296] Seq ID NO:41
represents amino acid sequence of VL, 002-H06 [0297] Seq ID NO:42
represents amino acid sequence of VL, 002-H08 [0298] Seq ID NO:43
represents amino acid sequence of VL, 006-H07 [0299] Seq ID NO:44
represents amino acid sequence of VL, 001-E06 [0300] Seq ID NO:45
represents amino acid sequence of VL, 005-C04 [0301] Seq ID NO:46
represents nucleic acid sequence VH, 006-H08 [0302] Seq ID NO:47
represents nucleic acid sequence VH, 002-H06 [0303] Seq ID NO:48
represents nucleic acid sequence VH, 002-H08 [0304] Seq ID NO:49
represents nucleic acid sequence VH, 006-H07 [0305] Seq ID NO:50
represents nucleic acid sequence VH, 001-E06 [0306] Seq ID NO:51
represents nucleic acid sequence VH, 005-C04 [0307] Seq ID NO:52
represents nucleic acid sequence VL, 006-H08 [0308] Seq ID NO:53
represents nucleic acid sequence VL, 002-H06 [0309] Seq ID NO:54
represents nucleic acid sequence VL, 002-H08 [0310] Seq ID NO:55
represents nucleic acid sequence VL, 006-H07 [0311] Seq ID NO:56
represents nucleic acid sequence VL, 001-E06 [0312] Seq ID NO:57
represents nucleic acid sequence VL, 005-C04 [0313] Seq ID NO:58
represents amino acid sequence of VH, HE06642, Novartis
(WO2008/22295) [0314] Seq ID NO:59 represents amino acid sequence
of VH, XHA06642, Novartis (WO2008/22295) [0315] Seq ID NO:60
represents amino acid sequence of VH, XHA06983, Novartis
(WO2008/22295) [0316] Seq ID NO:61 represents amino acid sequence
of VL, HE06642 [0317] Seq ID NO:62 represents amino acid sequence
of VL, XHA06642 Novartis (WO2008/22295) [0318] Seq ID NO:63
represents amino acid sequence of VL, XHA06983 Novartis
(WO2008/22295) [0319] Seq ID NO:64 represents nucleic acid sequence
VH, HE06642 [0320] Seq ID NO:65 represents nucleic acid sequence
VH, XHA06642 Novartis (WO2008/22295) [0321] Seq ID NO:66 represents
nucleic acid sequence VH, XHA06983 Novartis (WO2008/22295) [0322]
Seq ID NO:67 represents nucleic acid sequence VL, HE06642 [0323]
Seq ID NO:68 represents nucleic acid sequence VL, XHA06642,
Novartis (WO2008/22295) [0324] Seq ID NO:69 represents nucleic acid
sequence VL, XHA06983, Novartis (WO2008/22295) [0325] Seq ID NO:70
represents human ECD_PRLR, amino acid position 1-210, 51 domain
1-100 (51 domain construct 1-102), S2 domain 101-210 [0326] Seq ID
NO:71 represents CDS human ECD_PRLR, nucleotide position 1-630
[0327] Seq ID NO:72 represents murine ECD_PRLR, amino acid position
1-210 [0328] Seq ID NO:73 represents CDS murine ECD_PRLR,
nucleotide position 1-630
EXAMPLES
Example 1
[0329] Isolation of target-specific antibodies from human antibody
phage display libraries To isolate a panel of antibodies able to
neutralize the activity of human PRLR, three human antibody phage
display libraries, expressing Fab and scFv fragments, were
investigated in parallel. The target used for the library panning
was the soluble extracellular domain (ECD) of the prolactin
receptor (human prolactin receptor amino acids 25-234) prepared as
described above in WO08/022295 (Novartis). Alternative targets were
the ECD of PRLR C-terminally linked to six histidines or to a human
IgG1-Fc domain via the linker with the amino acid sequence
"isoleucine-glutamate-glycine-arginine-methionine-aspartate".
[0330] Selection of target-specific antibodies from phage display
was carried out according to methods described by Marks et al.
(Methods Mol Biol. 248:161-76, 2004). Briefly, the phage display
library was incubated with 50 pmols of the biotinylated ECD at room
temperature for 1 hr and the complex formed was then captured using
100 .mu.l of Streptavidin beads suspension (Dynabeads .RTM. 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.
[0331] Microcultures were grown to an OD.sub.60O=O.6 at which point
expression of soluble antibody fragment was induced by addition of
1 mM IPTG following overnight culture in a shaker incubator at
30.degree. C. Bacteria were spun down and periplasmic extract was
prepared and used to detect antibody binding activity to ECD
immobilized on 96-well microplates (96-well flat bottom lmmunosorb
plates, Nunc) following standard ELISA protocol provided by the
microplate manufacturer.
[0332] The affinities of the anti-Prolactin Receptor (PRLR)
antibodies for binding to the recombinant extracellular domain
(ECD) were estimated using the Biacore.RTM. 2000 and used for
affinity ranking of antibodies.
Example 2
Quantitative Analysis of Prolactin and Prolactin Receptor Gene
Expression by Real-Time Taqman PCR Analysis In Eu- and Ectopic
Endometrium and Endometriotic Lesions from Patients and Healthy
Controls
[0333] 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. We analyzed the expression of PRL
and the PRLR in the endometrium from healthy women and in
endometrium and endometriotic lesions from patients by using
quantitative real-time Taqman PCR analysis. The expression of
prolactin and its receptor was clearly upregulated in endometriotic
lesions compared to healthy endometrium or endometrium derived from
patients.
[0334] Results are shown in FIGS. 1 and 2.
[0335] These findings imply that autocrine prolactin signaling
plays a role in the development and maintenance of endometriosis
and adenomyosis uteri (endometriosis interna, a form of
endometriosis restricted to the uterus).
Example 3
Analysis of Prolactin Receptor Expression in Human Tissues by
Northern Blot
[0336] RNA was isolated from different rat tissues and transferred
to a nylon membrane after gel electrophoresis. The membranes were
successively hybridized with radioactive labelled cDNAs for the rat
prolactin receptor or .beta.-actin (as loading control), washed,
and exposed to film. The bands correspond to the mRNAs for the rat
prolactin receptor and .beta.-actin. The results shown in FIG. 3
indicate a strong expression of the prolactin receptor in the
placenta, the prostate, the ovary and the adrenal gland.
Example 4
Regulation of Prolactin Receptor Protein Expression in Rat
Prostate--Influence of Castration and Hormonal Treatments
[0337] Rats were either castrated or remained intact. Intact
animals were treated daily for 14 days with vehicle (intact), DHT
(3 mg/kg), or E2 (0.4 mg/kg). Afterwards prostates were isolated
from animals of all treatment groups and protein extracts were
prepared. Protein extracts were separated by gel electrophoresis
and transferred to a membrane. The prolactin receptor was detected
using the commercially available antibody MA610 (Santa Cruz
Biotechnology). The results are shown in FIG. 4 and indicate the
hormonal regulation of the prolactin receptor in the rat
prostate.
Example 5
[0338] Inhibition of Prolactin-Induced Proliferation Of Baf3 Cells
(Stably Transfected with Human Prolactin Receptor) by Neutralizing
Prolactin Receptor Antibodies and Unspecific Control Antibodies
[0339] To analyze the in vitro efficacy of the neutralizing PRLR
antibodies, the inhibition of prolactin-activated cellular
proliferation of BaF3 cells was used. The cells were stably
transfected with human PRLR and were routinely cultured in RPMI
containing 2 mM glutamine in the presence of 10% FCS and 10 ng/ml
of human prolactin. After six hours of starvation in prolactin-free
medium containing 1% FCS, cells were seeded into 96-well plates at
a density of 10000 cells per well. Cells were stimulated with 20
ng/ml prolactin and coincubated with increasing doses of
neutralizing PRLR antibodies for two days. Cellular proliferation
was analyzed using a CellTiter-Glo Luminescent Cell Viability Assay
(Promega). Dose-response curves for the inhibition of
prolactin-stimulated cellular growth were generated and IC.sub.50
values calculated. As negative control, stimulation with an
unspecific control antibody was used.
[0340] The dose-response curves and IC.sub.50 values are depicted
in FIG. 5. The unspecific antibody did not inhibit the
proliferation of BaF cells stably expressing the human PRLR,
whereas the specific antibodies blocked cell proliferation and
exhibited different potencies. Neutralizing antibody 006-H08 showed
the highest potency in this readout paradigm.
Example 6
Inhibition of Prolactin-Induced Rat Lymphoma Cell Proliferation by
Specific and Unspecific Antibodies
[0341] The in vitro efficacy of the neutralizing PRLR antibodies
was also tested using inhibition of prolactin-dependent rat
lymphoma cell (Nb2-11 cells) proliferation. Nb2-11 cells were
routinely grown in RPMI containing 10% FCS and 10% horse serum.
Before starting cellular growth assays, cells were grown for 24
hours in the same medium containing 1% FCS instead of 10% FCS.
Afterwards, cells were seeded in 96-well plates in FCS-free medium
at a density of 10000 cells per well. Cells were stimulated with 10
ng/ml human prolactin in the presence or absence of increasing
doses of neutralizing PRLR antibodies or control antibodies for 2
days. Afterwards cellular proliferation was assessed using a
CellTiter-Glo Luminescent Cell Viability Assay (Promega).
Dose-response curves and IC.sub.50 values are depicted in FIG. 6.
The unspecific antibody and antibody XHA06.983, that does not bind
the rat PRLR, did not block Nb2-11 cell proliferation. XHA06.642
which binds the rat PRLR blocked Nb2-11 cell proliferation.
Example 7
Inhibition of Prolactin-Induced STAT5 Phosphorylation in T47D Cells
by Neutralizing Prolactin Receptor Antibodies
[0342] To analyze the in vitro efficacy of the neutralizing PRLR
antibodies in an additional readout, the inhibition of STAT5
phosphorylation in human T47D cells treated with prolactin was
used. T47D cells were grown in RPMI containing 10% FCS and 2 mM
glutamine. Cells were seeded on 24-well plates at a density of
0.5.times.10.sup.5 cells per well. The next day, cells were starved
for 1 h in serum free RPMI. Afterwards cells were incubated with or
without different doses of neutralizing PRLR antibodies or
unspecific control antibody in the absence or presence of 20 ng/ml
human prolactin for 30 min. Afterwards cells were rinsed and lysed
in 70 .mu.l of lysisbuffer. Lysates were centrifuged and the
supernatant was frozen at -80.degree. C. Extracts were analyzed
using Western blot (anti-pSTAT5A/B antibody from upstate 07-586,
1:1000 diluted). As loading control the stripped blots were
incubated with anti-beta tubulin antibody (ab7287, 1:500 diluted).
Results are shown in FIG. 7. With the exception of the unspecific
FITC antibody, all neutralizing PRLR antibodies blocked STAT5
phosphorylation in human T47D cells dose-dependently. All tested
antibodies bound to the human PRLR with high affinity.
Example 8
[0343] Inhibition of Luciferase Reporter Gene Activity in Hek293
Cells Stably Transfected with the Human PRLR--Analysis of
Neutralizing Prolactin Receptor Antibodies and Unspecific Control
Antibodies
[0344] To further analyze the in vitro efficacy of the neutralizing
PRLR antibodies, a reporter gene assay was used. HEK293HEK293 cells
stably transfected with the human PRLR were transiently transfected
with a luciferase reporter gene under the control of LHREs
(lactogenic hormone response elements) for 7 hours. Afterwards,
cells were seeded at a density of 20000 cells per well on a 96-well
plate (0.5% charcoal stripped serum, DMEM). The next day 300 ng/ml
human prolactin with and without increasing doses of neutralizing
PRLR antibodies or control antibodies was added. 24 hours later,
luciferase activity was determined. Results are depicted in FIG. 8.
In contrast to the unspecific antibody, 006-H08 and HE06.642
inhibited luciferase activity in HEK293 cells stably transfected
with the human PRLR.
Example 9
[0345] Inhibition of Luciferase Reporter Gene Activity in Hek293
Cells Stably Transfected with the Murine PRLR--Analysis of
Neutralizing Prolactin Receptor Antibodies and Unspecific Control
Antibodies
[0346] To further analyze the in vitro efficacy of the neutralizing
PRLR antibodies on the murine prolactin receptor, a reporter gene
assay was used. HEK293 cells stably transfected with the murine
PRLR were transiently transfected with a luciferase reporter gene
under the control of LHREs (lactogenic hormone response elements)
for 7 hours. Afterwards, cells were seeded at a density of 20000
cells per well on a 96-well plate (0.5% charcoal stripped serum,
DMEM). The next day 200 ng/ml human prolactin with and without
increasing doses of neutralizing PRLR antibodies or control
antibodies was added. 24 hours later, luciferase activity was
determined. Results are depicted in FIG. 9. Whereas the antibody
005-C04 (closed triangles) exhibits high activity (IC.sub.50
value=3 nM), the antibody HE06.642 (closed circles) does not show
activity up to 330 nM. The unspecific control antibody (open
circles) is completely inactive. In contrast to the Novartis
antibody HE06.642, the antibody 005-C04 is able to block murine
PRLR-mediated signaling.
Example 10
[0347] Inhibition of Prolactin-Induced Proliferation of BaF3 Cells
(Stably Transfected with the Murine Prolactin Receptor) by
Neutralizing Prolactin Receptor Antibodies and Unspecific Control
Antibodies
[0348] To analyze the in vitro efficacy of the neutralizing PRLR
antibodies, the inhibition of prolactin-activated cellular
proliferation of Ba/F3 cells was used. The cells were stably
transfected with the murine PRLR and were routinely cultured in
RPMI containing 2 mM glutamine in the presence of 10% FCS and 10
ng/ml of human prolactin. After six hours of starvation in
prolactin-free medium containing 1% FCS, cells were seeded into
96-well plates at a density of 10000 cells per well. Cells were
stimulated with 40 ng/ml prolactin and coincubated with increasing
doses of neutralizing PRLR antibodies for two days. Cellular
proliferation was analyzed using a CellTiter-Glo Luminescent Cell
Viability Assay (Promega). Dose-response curves for the inhibition
of prolactin-stimulated cellular growth were generated and
IC.sub.50 values calculated. As negative control, stimulation with
an unspecific control antibody was used.
[0349] The dose-response curves and IC.sub.50 values are depicted
in FIG. 10. The unspecific control antibody (closed squares) was
inactive at the murine PRLR. There was only limited inhibition of
murine PRLR activation by the antibodies HE06.642, 001-E06, and
001_D07. Only antibody 005-C04 completely blocked murine PRLR
activation.
Example 11
Contraceptive Effect of Neutralizing Prolactin Receptor Antibody
IgG1 005-C04 in Mice
[0350] To test the influence of neutralizing prolactin receptor
antibodies on fertility in mice, 12 week old female and male NMRI
mice were mated for 7 days (day 0-day 7). Female mice were treated
on days -3, 0, 3, and 6 with an intraperitoneal injection of either
phosphate-buffered saline, unspecific IgG1 control antibody
(anti-FITC, 10 mg/kg), or the neutralizing IgG1 antibody 005-C04
(=IgG1 005-C04) at concentrations of 10 or 30 mg per kg body weight
dissolved in phosphate buffered saline. 10 females were used in
each experimental group. Each male was mated with two females, one
of the females was from a negative control group treated with
either phosphate-buffered saline or unspecific antibody, the other
female was treated with specific neutralizing antibody. Matings, in
which the male did not produce at least one pregnant female, were
excluded from data evaluation. Readout parameters were mean litter
size and pregnancy rates (measured in %) calculated as litter
number per experimental group divided by the number of theoretical
possible litters within this group. Results are depicted in FIG.
11.
[0351] FIG. 11A shows the obtained pregnancy rates. Pregnancy rates
were as follows:
87.5% in the group of mice treated with phosphate buffered saline,
75% in the group of mice treated with the unspecific control
antibody (10 mg/kg), 100% in the group of mice treated with the
neutralizing PRLR antibody IgG1 005-C04 (10 mg/kg), and 0% in the
group of mice treated with the neutralizing PRLR antibody IgG1
005-C04 (30 mg/kg).
[0352] FIG. 11B shows the observed litter sizes for the different
experimental groups. Litter sizes were as follows: [0353] 10.9 mice
per litter in the group of mice treated with phosphate buffered
saline, [0354] 12.3 mice per litter in the group of mice treated
with the unspecific control antibody (10 mg/kg), [0355] 13 mice per
litter in the group of mice treated with the neutralizing PRLR
antibody IgG1 005-C04 (10 mg/kg), and [0356] 0 mice per litter in
the group of mice treated with the neutralizing PRLR antibody IgG1
005-C04 (30 mg/kg).
[0357] The results from this mating study demonstrate that the
neutralizing prolactin receptor antibody IgG1 005-C04 completely
prevented pregnancy in mice when tested at 30 mg/kg body
weight.
Example 12
Epitope Grouping
[0358] 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. Two antibodies recognizing the same or
overlapping epitopes cannot bind to the ECD-PRLR, whereas
antibodies with distinct epitopes are able to bind. The antibody
surface was regenerated with glycine, pH 2.8, to remove bound
proteins and then the process was repeated with other antibodies.
All combinations of antibodies were tested. Representative results
are shown in Table 7. The antibodies 006-H08, 002-H06, 002-H08,
006-H07 and XHA06983 competitively bound to each other on ECD-PRLR,
indicating that they target overlapping epitopes (epitope group 1,
table 6). In addition, the antibodies competitively bound to PRL,
which is also the case for 001-E06 (epitope group 2, table 6). This
antibody targets a different site of ECD-PRLR than the afore
mentioned ones. Finally, the antibody 005-C04 competitively bound
to HE06.642 and XHA06.642 without being competitive to PRL (epitope
group 3, table 6).
TABLE-US-00007 TABLE 7 Groups of antibodies which target
overlapping epitopes on the extracellular domain (ECD) of the human
prolactin receptor (PRLR) Competition to Antibody Epitope group
prolactin 006-H08 1 Yes 002-H06 1 Yes 002-H08 1 Yes 006-H07 1 Yes
001-E06 2 Yes 005-C04 3 No HE06.642 3 No XHA06.642 3 No XHA06.983 1
Yes
Example 13
Cross-Reactivity of Antibodies on Mouse and Human PRLR Expressed on
Cell Surfaces
[0359] In order to determine the binding characteristics of the
anti-PRLR antibodies on mouse and human PRLR expressed on cells,
binding was tested by flow cytometry on HEK293 cells stably
expressing the human and murine PRLR, respectively. The cells as
well as the parental HEK293 cell line without PRLR were harvested,
centrifuged and resuspended at approximately 5.times.10.sup.6
cells/ml in 1.times.PBS 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 .mu.l/well). For secondary antibody
and autofluorescence controls, 50 .mu.l FACS buffer was added to
appropriate wells. 50 .mu.l of cell suspension was added to each
sample well. Samples were incubated at 4.degree. C. for one hour,
washed twice with cold FACS buffer and resuspended in FACS buffer
containing PE-conjugated goat anti-human IgG at a 1:100 dilution.
Following a 30 min incubation at 4.degree. C., cells were washed
twice with cold FACS buffer, resuspended in FACS buffer containing
1 .mu.g/ml propidium iodide (Invitrogen, San Diego, Calif.) and
analyzed by flow cytometry. As shown in FIG. 13, the antibodies
005-C04 and 001-E06 bound to human and murine PRLR on these cells,
while HE06.642 only bound to the human PRLR. This observation is
consistent with the finding reported in example 9 about the missing
efficacy of HE06.642 in the murine PRLR-dependent luciferase
reporter gene assay. Although 005-C04 and HE06.642 competitively
bound to human PRLR, the different binding properties of both
antibodies with respect to the murine PRLR indicate differences in
their epitope specificity.
Example 14
[0360] Inhibitory Activity of Fab and scFv Antibodies on Cellular
Signaling Cascades
[0361] To functionally characterize the activity of the Fab and
scFv screening hits on the PRLR-triggered signaling cascade, the
inhibition of phosphorylation on PRLR itself, and on the
transcriptional regulators ERK1/2 and STAT5 in human T47D cells
treated with prolactin was measured. T47D cells were grown in RPMI
containing 2 mM L-glutamine, 10% charcoal stripped FBS and
insulin-transferrin-selenium-A (Gibco). Cells were seeded on 6 well
plates or 96-well plates at a density of 1.5.times.10.sup.6 cells
per well. The next day, growth medium was renewed. On the third,
day cells were starved for 1 hour in serumfree RPMI. Afterwards
cells were incubated with or without different doses of
neutralizing PRLR antibodies or unspecific control antibody in the
presence of 500 ng/ml human prolactin for 5 min. Afterwards cells
were rinsed and lysed in lysis buffer. Lysates were centrifuged and
the supernatants were frozen at -80.degree. C. Samples were tested
by ELISA according to the DuoSet IC "Human Phospho-Prolactin R" kit
(R&D Systems) for measurement of PRLR phosphorylation,
according to the PathScan Phospho-STAT5 (Tyr694) Sandwich ELISA kit
(Cell Signaling Technology; #7113) for measurement of STAT5
phosphorylation and according to the Phospho-ERK1/ERK2 kit (R&D
Systems) for measurement of ERK1/2 phosphorylation. Table 8
provides an overview about the antagonistic activity of a selection
of screening hits in Fab or scFv format at a fixed dose of 7.5
.mu.g per ml.
TABLE-US-00008 TABLE 8 Antagonistic activity of a selection of
screening hits on the phosphorylation of PRLR, ERK1/2 and STAT5 as
determined by ELISAs on cell lysates of the human breast cancer
cell line T47D Inhibition of phosphorylation in % at a fixed
antibody dose (7.5 .mu.g/ml) Antibody PRLR ERK1/2 STAT5 006-H08*
100 100 100 002-H06.degree. 92 86 72 002-H08.degree. 100 100 98
006-H07* 88 85 73 001-E06.degree. 63 45 36 Negative control 2 9 0 *
scFv format, .degree.Fab format
Example 15
Neutralizing PRLR Antibodies Inhibit Lactation in Mice
[0362] Adult NMRI females were mated with NMRI males. On postpartal
day 1, litter size was adjusted to 8 mice per lactating mother. The
weight of the offspring was determined daily in the morning
starting on postpartal day 1. Lactating mothers remained either
untreated (closed circles in FIG. 14A,B) or were treated
intraperitoneally with either unspecific antibody (10 mg/kg body
weight; open circles in FIG. 14A,B), or with neutralizing PRLR
antibody 005-C04 containing murine IgG2a constant domains (=IgG2a
005-C04; 10 mg/kg, closed triangles in FIG. 14A, B) or with
neutralizing PRLR antibody IgG2a 005-C04 (30 mg/kg, open triangles
in FIG. 14A, B). Group size was 5-6 lactating mothers per
experimental group. Mothers were treated with specific or
unspecific control antibodies on postpartal day 1, 3, 6, 9, 10, and
12 (indicated with arrows in FIG. 14A, B). The results are depicted
in FIG. 14. FIG. 14A shows for each postpartal day the daily litter
weight gain expressed as percentage of the respective litter weight
on day 1. From postpartal day 8 onwards there is a significant
difference in litter weight gain between offspring from mothers
treated with neutralizing PRLR antibodies and offspring from
mothers that remained untreated or received unspecific control
antibodies. Due to ethical reasons several litters had to be killed
on postpartal day 10 in the experimental group of mothers receiving
the highest dose of the neutralizing PRLR antibody. In FIG. 14B the
results are depicted in a different way. The differential litter
weight gain from day to day is depicted and expressed as percentage
of the litter weight on postpartal day 1. Basically FIG. 14B shows
the slope of the graphs depicted in FIG. 14A. The differential
daily increase in litter weight oscillates around 30% of the
starting litter weight on postpartal day 1 for litters from
untreated mothers or mothers treated with the unspecific antibody.
There is a significant severe reduction in daily litter weight
increase in litters from mothers treated with the neutralizing PRLR
antibody at 30 mg/kg body weight from day 7 onwards (*p<0.05;
***p<0.005 vs. litters from mothers treated with unspecific
antibody). From postpartal day 11 onwards, daily litter weight
increase is significantly diminished also in litters from mothers
treated with the neutralizing PRLR antibody at 10 mg/kg if compared
to litters from mothers treated with unspecific control antibodies
(p<0.05 vs. litters from mothers treated with unspecific
antibody). In conclusion, there are dose-dependent effects of the
neutralizing PRLR antibody IgG2a 005-C04 on lactation inhibition.
FIG. 14C shows histological sections of the mammary glands from
lactating mothers of the different experimental groups. Mammary
glands of untreated mothers and mothers treated with unspecific
control antibodies are filled with ducts producing milk. In
contrast, there are signs of mammary gland involution in mothers
treated with the neutralizing PRLR antibody IgG2a 005-C04. Black
arrows in FIG. 14C point to fatty islands in the mammary gland
tissue (see dose-dependent effect of the specific antibody IgG2a
005-004 on the extent of mammary gland involution (FIG. 14C)). In
addition, the expression of the major milk proteins
bet.alpha.-casein (Csn-2), whey acidic protein (WAP), and IGF-1 in
the mammary glands of mothers from the different experimental
groups were analyzed (FIG. 14D). Gene expression was normalized to
the expression of TATA-box binding protein (TBP). The neutralizing
PRLR antibody IgG2a 005-C04 dose-dependently decreased milk protein
expression whereas the unspecific antibody (10 mg/kg) was without
any significant effect.
[0363] The neutralizing PRLR antibody IgG2a 005-C04
dose-dependently blocked lactation and lead to mammary gland
involution in lactating mice demonstrating its usefullness for
lactation inhibition.
Example 16
Neutralizing PRLR Antibodies are Suitable for the Treatment of
Benign Breast Disease
[0364] An activating PRLR mutation or local or systemic
hyperprolactinemia can provoke benign breast disease. Therefore, a
hyperprolactinemic mouse model to induce enhanced proliferation in
the mammary gland (hallmark of the most severe forms of benign
breast disease) was employed. On day 0, 12 week old female Balb/c
mice received a pituitary isograft under the kidney capsule or
remained unoperated. Pituitary isografted mice remained untreated
or were treated intraperitoneally with either unspecific antibody
(10 mg/kg), neutralizing PRLR antibody 005-C04 in IgG1 format
(=IgG1 005-C04; 10 mg/kg), or neutralizing PRLR antibody IgG1
005-C04 (30 mg/kg) on day 0, 3, 7, 11, and 15. Experimental group
size was 8-10 animals. On day 17 after pituitary transplantation
mice were sacrificed. Two hours before death, animals received an
intraperitoneal injection of BrdU to monitor epithelial cell
proliferation. The left inguinal mammary gland was fixed in
Carnoy's solution and mammary gland whole mounts were prepared and
stained with Carmine alaune (FIG. 15A). The right inguinal mammary
gland was fixed in 4% phosphate-buffered formaline overnight.
Mammary glands were subsequently embedded in paraffin and BrdU
immunostainings were performed as described previously
(Endocrinology 149(8):3952-3959;2009). In addition, a pSTAT5
immunostaining was performed (anti pSTAT5 antibody from abcam,
ab32364, diluted 1:60) to monitor the inhibition of PRLR-mediated
signaling in response to treatment with neutralizing PRLR
antibodies. FIG. 15A shows magnifications of mammary gland whole
mounts from the different experimental groups. Mammary glands of
adult mice that did not receive a pituitary show ducts and endbuds,
whereas there is extreme side branching and formation of alveolar
structures in mice receiving a pituitary isograft. Treatment with
the unspecific antibody (10 mg/kg) did not inhibit side branching
and formation of alveolar structures. In contrast, treatment with
the neutralizing antibody IgG1 005-C04 at 10 mg/kg body weight
leads to complete inhibition of side branching in 8 out of 10
animals receiving a pituitary isograft and treatment with IgG1
005-C04 at 30 mg/kg completely inhibits side branching in 9 out of
9 animals receiving a pituitary isograft. Histological analysis and
BrdU immunostaining are depicted in FIG. 15B. Pituitary isografting
leads to epithelial hyperplasia that is not inhibited by treatment
with the unspecific antibody, whereas there is no epithelial
hyperplasia in mice harbouring a pituitary isograft and treated
with the neutralizing PRLR antibody at a dose of 10 or 30 mg/kg
body weight. Some of the BrdU-positive cells, reflecting cells in
the S-phase of the cell cylcle which are going to divide, are
indicated by white arrows in FIG. 15B. Mice treated with the
neutralizing antibody
[0365] IgG1 005-C04 (30 mg/kg body weight) showed almost complete
inhibition of epithelial cell proliferation in mammary glands. Some
of the cells positive for phospho-STAT5 are indicated by white
arrows in FIG. 15C. Treatment with 30 mg/kg IgG1 005-C04 lead to
complete inhibition of STAT5 phosphorylation, indicating complete
blockade of PRLR-mediated signaling.
[0366] The results from FIG. 15A, B, and C demonstrated that
neutralizing PRLR antibodies are suitable for the treatment of
mastopathia, a benign proliferative disease of the mammary gland.
Neutralizing PRLR antibodies inhibit mammary epithelial cell
proliferation and activation of phospho-STAT5.
Example 17
[0367] Treatment of Benign Prostate Hyperplasia with Neutralizing
PRLR Antibodies
[0368] Benign prostate hyperplasia was established in male Balb/c
mice by grafting of two pituitaries under the kidney capsule at the
age of 8 weeks. A control group remained unoperated. Mice receiving
pituitary isografts remained untreated or received intraperitoneal
injections of either an unspecific antibody (10 mg/kg), or the
neutralizing PRLR antibody 005-C04 containing murine IgG2a constant
domains (=IgG2a 005-C04) at doses of 10 and 30 mg/kg body weight.
Antibody injections were performed starting on the day of pituitary
transplantation (=day 0), and on day 3, day 7, day 11, day 15, day
18, day 22, and day 25 after pituitary transplantation. Mice were
sacrificed on day 28. The relative weight of the ventral prostate
was determined. Results are depicted in FIG. 16. Pituitary
isografting resulted in an increase in relative prostate weight.
Treatment with 10 mg/kg and 30 mg/kg neutralizing PRLR antibody
IgG2a 005-C04 reduced prostate weight whereas treatment with
unspecific control antibody was without any effect. Neutralizing
PRLR antibodies are therefore suitable for the treatment of benign
prostate hyperplasia.
[0369] On day 18 after pituitary isografting it became evident that
hair growth was diminished in animals receiving pituitary
isografts. Neutralizing PRLR anibodies stimulated hair growth under
hyperprolactinemic conditions. Representative photographs are shown
in FIG. 17. Therefore neutralizing PRLR antibodies can be used for
the treatment of hyperprolactinemic hair loss.
Example 18
Effect of Neutralizing PRLR Antibodies on Hair Growth
[0370] The dorsal hair of 8 weeks old male and female C57BL/6 mice
was removed using electric shawers as descibed previously (British
Journal of Dermatology 2008;159:300-305). Hyperprolactinemia was
induced in some groups by pituitary isografting under the kidney
capsule, animals in the remaining groups were normoprolactinemic.
Animals were treated with specific PRLR antibodies (IgG2a 005-C04)
or unspecific control antibodies (30 mg/kg, intraperitoneally) once
weekly (starting on day 0 which is the day of pituitary
isografting). Subsequent antibody injections were performed on days
7 and 14. After three weeks, the regrown hair was visible as dark
on the pinkish-white shaved skin, and the percentage of the shaved
area that became dark was measured. Female mice were killed 15 days
after shaving and male mice were sacrificed 18 days after
shaving.
[0371] The following experimental groups were used (group size was
6 mice): [0372] 1. shaved females [0373] 2. shaved females with
pituitary isograft [0374] 3. shaved females with pituitary
isograft+30 mg/kg unspecific antibody IgG2a 005-C04 once weekly
[0375] 4. shaved females with pituitary isograft+30 mg/kg specific
antibody once weekly [0376] 5. shaved females+30 mg/kg unspecific
antibody once weekly [0377] 6. shaved females+30 mg/kg specific
antibody once weekly [0378] 7. shaved males [0379] 8. shaved males
with pituitary isograft [0380] 9. shaved males with pituitary
isograft+30 mg/kg unspecific antibody once weekly [0381] 10. shaved
males with pituitary isograft+30 mg/kg specific antibody IgG2a
005-C04 once weekly [0382] 11. shaved males+30 mg/kg unspecific
antibody once weekly [0383] 12. shaved males+30 mg/kg specific
antibody once weekly
[0384] Representative pictures from animals of the different groups
are depicted in FIG. 18, the percentage of the area regrown with
hair is indicated in FIG. 18. Neutralising PRLR antibodies, but not
unspecific antibodies, stimulate hair regrowth under hyper- and
normoprolactinemic conditions in male and female mice. Neutralising
PRLR antibodies are therefore suitable to treat hair loss in women
and men under hyper- and normoprolactinemic conditions.
Example 19
Inhibition of Enhanced Mammary Epithelial Cell Proliferation by
Neutralizing PRLR Antibodies
[0385] To test the effect of neutralizing PRLR antibodies on
enhanced mammary epithelial cell proliferation activated by
combined hormone therapy (i.e. estrogen plus progestin therapy) a
previously described mouse model that allowed for the
quantification of proliferative effects in the uterus and the
mammary gland was employed (Endocrinology 149:3952-3959,2008). 6
week old C57BL/6 female mice were ovariectomized. 2 weeks after
ovariectomy, animals were treated subcutaneously with daily
injections of either vehicle (ethanol/arachisoil 10%/90%) or 100 ng
estradiol plus 100 mg/kg progesterone for two weeks. Animals were
treated once weekly with intraperitoneal injections of neutralizing
PRLR antibodies (10 mg/kg and 30 mg/kg) in the murine IgG2a format
or unspecific antibody (30 mg/kg) for three weeks. Autopsy was
performed on day 36 after ovariectomy. Two hours before death
animals received an intraperitoneal injection of BrdU dissolved in
phosphate buffered saline (70 mg/kg body weight). The proximal 2/3
of the right inguinal mammary gland was analyzed for mammary
epithelial cell proliferation (BrdU immunostaining) described
previously (Endocrinology 149:3952-3959,2008).
[0386] The experiment comprised the following groups: [0387] 1.
ovariectomized animals treated with vehicle [0388] 2.
ovariectomized animals treated with 100 ng estradiol [0389] 3.
ovariectomized animals treated with 100 ng estradiol (E) and 100
mg/kg progesterone (P) [0390] 4. ovariectomized animals treated
with E+P and 10 mg/kg specific antibody 005-C04 [0391] 5.
ovariectomized animals treated with E+P and 30 mg/kg specific
antibody 005-C04 [0392] 6. ovariectomized animals treated with E2+P
and 30 mg/kg unspecific control antibody
[0393] The results are shown in FIG. 19. The absolute number of
proliferating ductal epithelial cells within 4 cross-sections of
the mammary gland was evaluated. The medians are depicted as
horizontal bars. Epithelial cell proliferation in ovariectomized,
vehicle treated mice is rather low. Estradiol treatment leads to
some stimulation of epithelial cell proliferation, maximal mammary
epithelial cell proliferation is observed under estrogen plus
progesterone treatment (FIG. 19). Treatment with neutralising
prolactin receptor antibody 005-C04 but not with unspecific control
antibody leads to a dose-dependent decrease in mammary epithelial
cell proliferation almost back to estradiol-only levels.
[0394] Neutralising PRLR antibodies are therefore suitable to treat
enhanced mammary epithelial cell proliferation under combined
hormone therapy, i.e. estradiol plus progesterone treatment.
Example 20
[0395] Treatment of Adenomyosis Uteri (=Endometriosis Interna) in
SHN Mice with Neutralizing PRLR Antibodies
[0396] To test the efficacy of neutralizing PRLR antibodies in
endometriosis, the adenomyosis uteri model in SHN mice relying on
systemic hyperprolactinemia was employed (Acta anat.
116:46-54,1983). Hyperprolactinemia in SHN mice was induced by
pituitary isografting under the kidney capsule of 7 weeks old
female mice (Acta anat. 116:46-54,1983). Neutralizing PRLR
antibodies (10 mg/kg or 30 mg/kg) or unspecific antibodies (30
mg/kg) were administered intraperitoneally starting one week after
pituitary isografting. The infiltration of the uterine muscular
layer by glandular tissue was assessed as described previously
(Laboratory Animal Science 1998,48:64-68). Treatment with the
antibodies was performed for 9 weeks once and twice weekly by
intraperitoneal injections. At autopsy (day 70 after pituitary
transplantation), uteri were fixed overnight in buffered 4%
formalin and embedded in paraffin. The degree of adenomyosis
(=endometriosis interna) was assessed as follows: [0397] Grade 0=no
adenomyosis [0398] Grade 0.5=the inner layer of the myometrium
looses its concentric orientation [0399] Grade 1=endometrial glands
invading the inner layer of the myometrium [0400] Grade
2=endometrial glands between the inner and outer layer of the
uterine myometrium [0401] Grade 3=endometrial glands invading the
outer layer of the uterine myometrium [0402] Grade 4=endometrial
glands outside of the outer layer of the uterine myometrium
[0403] The experiment comprised the following experimental groups:
[0404] 1. Animals without pituitary transplantation, i.e.
normoprolactinemic mice [0405] 2. Animals with pituitary
transplantation, i.e. hyperprolactinemic mice [0406] 3. Animals
with pituitary transplantation, treated with unspecific control
antibody once weekly at a dose of 30 mg/kg [0407] 4. Animals with
pituitary transplantation, treated with unspecific control antibody
twice weekly at a dose of 30 mg/kg [0408] 5. Animals with pituitary
transplantation, treated with the neutralizing prolactin receptor
antibody 005-C04 in the murine IgG2a format once weekly at a dose
of 10 mg/kg [0409] 6. Animals with pituitary transplantation,
treated with the neutralizing prolactin receptor antibody 005-C04
in the murine IgG2a format twice weekly at a dose of 10 mg/kg
[0410] 7. Animals with pituitary transplantation, treated with the
neutralizing prolactin receptor antibody 005-C04 in the murine
IgG2a format once weekly at a dose of 30 mg/kg [0411] 8. Animals
with pituitary transplantation, treated with the neutralizing
prolactin receptor antibody 005-C04in the murine IgG2a format twice
weekly at a dose of 30 mg/kg
[0412] The results are depicted in FIG. 20. The scores for each
animal in each treatment group are given individually and the
medians for each treatment group are shown as horizontal bars.
Normoprolactinemic mice develop endometriosis interna to some
degree (median disease score=0.25). Hyperprolactinemia due to
pituitary isografting enhances the disease score and more animals
suffer from the disease (median disease score=2.5). Whereas
treatment with 30 mg/kg unspecific antibody once or twice weekly
had no influence on the disease, treatment with specific
neutralizing antibodies shows a dose-dependent decrease in the
disease score. Notably, all animals receiving either 10 or 30 mg/kg
specific antibody twice weekly were completely cured and their
disease score was significantly lower than the disease score of
normoprolactinemic mice (FIG. 20). Neutralising PRLR antibodies are
therefore suitable to treat endometriosis interna (=adenomyosis
uteri) and endometriosis externa in women.
Sequence CWU 1
1
7318PRTHomo sapiens 1Phe Asp Asp Tyr Gly Met Ser Trp1 528PRTHomo
sapiens 2Phe Ala Asn Tyr Gly Leu Thr Trp1 538PRTHomo sapiens 3Phe
Ser Ser Tyr Gly Met His Trp1 548PRTHomo sapiens 4Phe Glu Asp His
Gly Met Ser Trp1 558PRTHomo sapiens 5Phe Ser Ser Tyr Trp Met Ser
Trp1 568PRTHomo sapiens 6Phe Ser Ser Tyr Trp Met His Trp1
5719PRTHomo sapiens 7Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val1 5 10 15Lys Gly Arg819PRTHomo sapiens 8Ala Val
Ile Ser Phe Asn Gly Asp Lys Lys Tyr Tyr Ala Asp Ser Val1 5 10 15Lys
Gly Arg919PRTHomo sapiens 9Ser Gly Val Ser Trp Asn Gly Ser Arg Thr
His Tyr Ala Asp Ser Val1 5 10 15Lys Gly Arg1020PRTHomo sapiens
10Ser Leu Ile Ser Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser1
5 10 15Val Lys Gly Arg 201119PRTHomo sapiens 11Ser Ser Val Ser Asp
Thr Gly Thr Asp Thr His Tyr Ala Asp Ser Val1 5 10 15Lys Gly
Arg1219PRTHomo sapiens 12Ser Asp Ile Ser Ser Ala Ser Ser Tyr Thr
Asn Tyr Ala Asp Ser Val1 5 10 15Lys Gly Arg1312PRTHomo sapiens
13Ala Ser Pro Leu Glu Ser Pro Val Ala Phe Asp Ile1 5 10149PRTHomo
sapiens 14Cys Ala Arg Gly Gly Asp Phe Asp Tyr1 51511PRTHomo sapiens
15Ala Thr Ser Leu Arg Ala Thr Ala Phe Asp Thr1 5 101616PRTHomo
sapiens 16Ala Lys Thr Pro Leu Ala Tyr Ser Ser Gly Trp Tyr Tyr Phe
Asp Tyr1 5 10 151711PRTHomo sapiens 17Ala Arg Gly Leu Asp Ala Arg
Arg Met Asp Tyr1 5 101813PRTHomo sapiens 18Ser Gly Ser Asn Ser Asn
Ile Gly Ser Asn Pro Val Asn1 5 101913PRTHomo sapiens 19Ser Gly Ser
Tyr Ser Asn Ile Gly Gly Asn Pro Val Asn1 5 102013PRTHomo sapiens
20Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Tyr1 5
102113PRTHomo sapiens 21Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala
Val Asn1 5 102213PRTHomo sapiens 22Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn Thr Val Asn1 5 102314PRTHomo sapiens 23Thr Gly Ser Ser Ser
Asn Ile Gly Ala Gly Tyr Val Val His1 5 10247PRTHomo sapiens 24Asp
Asn Asn Lys Arg Pro Ser1 5257PRTHomo sapiens 25Gly Asn Ser Asn Arg
Pro Ser1 5267PRTHomo sapiens 26Ser Asn Asn Gln Arg Pro Ser1
5277PRTHomo sapiens 27Arg Asn Tyr Gln Arg Pro Ser1 5287PRTHomo
sapiens 28Arg Asn Asn Gln Arg Pro Ser1 52911PRTHomo sapiens 29Cys
Gln Ser Tyr Asp Thr Gly Leu Ser Gly Trp1 5 103011PRTHomo sapiens
30Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser1 5 103112PRTHomo
sapiens 31Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Trp1 5
103211PRTHomo sapiens 32Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly
Trp1 5 103311PRTHomo sapiens 33Cys Ala Ala Trp Asp Asp Ser Leu Asn
Gly Trp1 5 1034119PRTHomo sapiens 34Gln Val Glu Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ser Pro Leu Glu Ser Pro Val Ala Phe Asp Ile Trp Gly Gln Gly 100 105
110Thr Met Val Ile Val Ser Ser 11535119PRTHomo sapiens 35Gln Val
Glu Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Tyr 20 25
30Gly Leu Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Phe Asn Gly Asp Lys Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Ser Pro Leu Glu Ser Pro Val Ala Phe Asp
Ile Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11536115PRTHomo sapiens 36Gln Val Glu Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Val Ser Trp Asn Gly
Ser Arg Thr His Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Leu Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser 11537119PRTHomo sapiens 37Gln Val Glu Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Glu Asp His 20 25 30Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Leu Ile Ser Trp
Asp Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser 50 55 60Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu65 70 75 80Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys
Ala Thr Ser Leu Arg Ala Thr Ala Phe Asp Thr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 11538123PRTHomo sapiens 38Gln Val
Glu Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Val Ser Asp Thr Gly Thr Asp Thr His Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Thr Pro Leu Ala Tyr Ser Ser Gly Trp
Tyr Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12039118PRTHomo sapiens 39Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Asp Ile Ser
Ser Ala Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Leu Asp Ala Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ser 11540112PRTHomo sapiens 40Asp
Ile Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn
20 25 30Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Tyr Asp Thr Gly Leu 85 90 95Ser Gly Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 11041112PRTHomo sapiens 41Gln Ser
Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Tyr Ser Asn Ile Gly Gly Asn 20 25
30Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 11042113PRTHomo sapiens 42Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Asp
Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp
Ser Ser Leu 85 90 95Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110Gln43112PRTHomo sapiens 43Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30Ala
Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 11044112PRTHomo sapiens 44Asp Ile Val Leu
Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile
Tyr Arg Asn Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65
70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
Leu 85 90 95Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln 100 105 11045113PRTHomo sapiens 45Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser
Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Val Val His
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr
Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu65 70 75
80Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser
85 90 95Leu Asn Gly Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110Gln46357DNAHomo sapiens 46caggtggaat tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg
ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attcccagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac actgccgtgt
attactgtgc gagtcccctt 300gaaagtcccg tcgcttttga tatctggggc
caagggacaa tggtcatcgt gagctca 35747357DNAHomo sapiens 47caggtggaat
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcgct aactacggcc tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtggcagtt atatcattta atggagacaa
aaaatattac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
actgccgtgt attactgtgc gagtcccctt 300gaaagtcccg tcgcttttga
tatctggggc caaggtaccc tggtcaccgt gagctca 35748345DNAHomo sapiens
48caggtggaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccagggaagg ggctggagtg ggtatcgggt gttagttgga atggcagtag
gacgcactat 180gcagactctg tgaagggccg actcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
actgccgtgt attactgtgc gagaggaggg 300gactttgact actggggcca
aggtaccctg gtcaccgtga gctca 34549357DNAHomo sapiens 49caggtggaat
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttgag gatcatggca tgagctgggt ccgccaagct
120ccagggaagg ggctggagtg ggtctctctt attagttggg atgatggaag
taataaatac 180tacgcagact ccgtgaaggg ccgattcacc atctccagag
acaattccaa gaacacgctg 240tatctgcaaa tgaacagcct gagagccgag
gacactgccg tgtattactg tgcgacttcc 300ctacgggcca cggcttttga
tacgtggggc caaggtacac tggtcaccgt gagctca 35750369DNAHomo sapiens
50caggtggaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagt agctattgga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcaagt gttagcgata ctggtactga
tactcattac 180gcagactccg tgaagggccg cttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
actgccgtgt attactgtgc aaaaacccct 300ctcgcatata gcagtggctg
gtactacttt gactactggg gccaaggtac cctggtcacc 360gtgagctca
36951354DNAHomo sapiens 51gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
agctactgga tgcactgggt ccgccaagct 120ccagggaagg ggctggagtg
ggtttcagac attagcagtg ctagtagtta cacaaactac 180gcagactcag
tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc
gaggggtttg 300gatgcgcgac ggatggacta ctggggccaa ggtaccctgg
tcaccgtgag ctca 35452336DNAHomo sapiens 52gatatcgtgc tgactcagcc
accctcagcg tctgggaccc ctgggcagag ggtcaccatc 60tcttgttctg gaagcaactc
caacatcgga agtaatcctg taaactggta tcagcagctc 120ccaggaacgg
cccccaaact cctcatctat gacaataata agcgaccctc aggggtccct
180gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag
tgggctccgg 240tccgaggatg aggctgatta ttactgccag tcctatgaca
ccggcctgag tggttgggtg 300ttcggcggag gaaccaagtt aaccgtccta ggtcag
33653336DNAHomo sapiens 53cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttccg gaagctactc caacatcggg
ggtaatcctg taaactggta tcagcagctc 120ccaggaacgg cccccaaact
cctcatctat ggtaacagca atcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgccag tcctatgaca gcagcctgag
tggttcggta 300ttcggcggag gaaccaagct gacggtccta ggtcag
33654339DNAHomo sapiens 54cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caatatcgga
agtaatgatg tatattggta tcagcagctc 120ccaggaacgg cccccaaact
cctcatctat gacaataata agcgaccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgccag tcctatgaca gcagcctgag
tggttcttgg 300gtgttcggcg gaggaaccaa gctgacggtc ctaggtcag
33955336DNAHomo sapiens 55cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcctgttctg gaagcagctc caacatcgga
aataatgctg taaactggta tcagcagctc 120ccaggaacgg cccccaaact
cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag
tggttgggtg 300ttcggcggag gaaccaagtt aaccgtccta ggtcag
33656336DNAHomo sapiens 56gatatcgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga
agtaatactg taaactggta tcagcagctc 120ccaggaacgg cccccaaact
cctcatctat agaaattatc agcgaccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgccag tcctatgaca gcagcctgag
tggttcggtg 300ttcggcggag gaaccaagtt aaccgtccta ggtcag
33657339DNAHomo sapiens 57cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacattggg
gcgggttatg ttgtacattg gtatcagcag 120ctcccaggaa cggcccccaa
actcctcatc tataggaata atcagcggcc ctcaggggtc 180cctgaccgat
tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc
240cggtccgagg atgaggctga ttattactgt gcagcatggg atgacagcct
gaatggttgg 300ctgttcggcg gaggaaccaa gttaaccgtc ctaggtcag
33958124PRTHomo sapiens 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Val Ser Ser Gly Gly
Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg His
Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr Tyr Ala Met Asp 100 105 110Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12059124PRTArtificial Sequencematurated PRLR antibody 59Glu Val Gln
Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly
Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40
45Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met
Tyr Tyr Cys 85 90 95Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr
Tyr Ala Met Asp 100 105 110Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 115 12060114PRTArtificial Sequencematurated PRLR antibody
60Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser
Phe 20 25 30Gly Met Gln Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala
Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys
Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Val Arg Ser Gly Arg Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val 100 105 110Ser Ser61113PRTHomo sapiens
61Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Lys Ser Val Ser Thr
Ser 20 25 30Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Glu Ser Gly
Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Pro Val Gln Ala Glu Asp Val Ala Thr Tyr
Tyr Cys Gln His Ser Gly 85 90 95Glu Leu Pro Pro Ser Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Ala62113PRTArtificial
Sequencematurated PRLR antibody 62Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Gly Ala Thr Ile Ser Cys
Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Thr Tyr Met His
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val
Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Gly 85 90 95Glu
Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Ala63108PRTHomo sapiens 63Ser Ile Val Met Thr Gln Thr Pro Lys
Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Gly Val Ser Asn Asp 20 25 30Val Ala Trp Phe Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Thr Arg
Tyr Thr Gly Val Pro Asp Arg Leu Thr Gly 50 55 60Ser Gly Tyr Gly Thr
Asp Phe Thr Phe Thr Ile Asn Thr Val Gln Ala65 70 75 80Glu Asp Leu
Ala Val Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Thr 85 90 95Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 10564372DNAHomo sapiens
64gaggtgcagc tcgtggagtc tggcggcgga ctggtgcagc ctggcggcag cctgagactg
60agctgcgccg tgagcggctt caccttcagc agctacggca tgagctgggt gcgccaggct
120cctggcaagg gactggaatg ggtggccacc gtgtccagcg gcggcaccta
cacctactac 180cccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc cagacaccgg 300ggcaactact acgccaccta
ctactatgcc atggactact ggggccaggg caccctggtg 360accgtgagct ca
37265372DNAArtificial Sequencematurated PRLR antibody 65gaggtgcagc
tcgtggagtc tggcggcgac ctggtgaagc ctggcggcag cctgaagctg 60tcctgcgccg
tgagcggctt caccttcagc agctacggca tgagctgggt gcgccagacc
120cccgacaaga gactggaatg ggtggcaacc gtgtctagcg gcggcaccta
cacctactac 180cccgacagcg tgaagggccg gttcaccatc agccgggaca
acgccaagaa caccctgtac 240ctgcagatgt ccagcctgaa gtccgaggac
agcgccatgt actattgcgc cagacatcgg 300ggcaactact acgccaccta
ctactatgcc atggactact ggggccaggg caccagcgtg 360accgtgagct ca
37266342DNAArtificial Sequencematurated PRLR antibody 66gacgtgcagc
tcgtggagtc tggcggcgga ctggtgcagc ctggcggaag ccggaaactg 60tcctgcgctg
ccagcggctt cgccttcagc agcttcggca tgcagtgggt gcgccaggcc
120cccgagaagg gcctggaatg ggtggcctac atcagcagcg gcagcagcac
catctactac 180gccgacaccg tgaagggccg gttcaccatc agcagagaca
accccaagaa taccctgttc 240ctgcagatga ccagcctgcg gagcgaggac
accgccatgt actactgcgt gcggagcggc 300agagactact ggggccaggg
caccagcgtg accgtgagct ca 34267339DNAHomo sapiens 67gatatcgtgc
tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgggccacc 60atcaactgca
aggccagcaa gtccgtgagc accagcggct acacctacat gcactggtat
120cagcagaagc ccggccagcc ccccaagctg ctgatctacc tggccagcaa
ccgggagagc 180ggcgtgcccg accggtttag cggcagcggc tccggcaccg
acttcaccct gaccatcagc 240cccgtgcagg ccgaggacgt ggccacctac
tactgccagc acagcggcga gctgcccccc 300agcttcggcc agggcaccaa
gctggaaatc aagcgggcc 33968339DNAArtificial Sequencematurated PRLR
antibody 68gatatcgtgc tgacccagag ccccgccagc ctggctgtgt ctctgggcca
gggcgccacc 60atcagctgcc gggccagcaa gtccgtgagc accagcggct acacctacat
gcactggtat 120cagcagaagc ccggccagcc ccccaagctg ctgatctacc
tggccagcaa cctggaaagc 180ggcgtgcccg ccagattcag cggcagcggc
tccggcaccg acttcaccct gaacatccac 240cccgtggagg aagaggacgc
cgccacctac tactgccagc acagcggcga gctgccccct 300agctttggcg
gcggaacaaa gctggaaatc aagcgggcc 33969324DNAArtificial
Sequencematurated PRLR antibody 69agcatcgtga tgacccagac ccccaagttc
ctgctggtgt ctgccggcga cagagtgacc 60atcacctgca aggccagcca gggcgtgagc
aacgacgtgg cctggttcca gcagaagccc 120ggccagagcc ccaagctgct
gatctacagc gccagcaccc ggtacaccgg cgtgcccgac 180agactgaccg
gctccggcta cggcaccgat ttcaccttca ccatcaacac cgtgcaggcc
240gaggacctgg ccgtgtactt ctgccagcag gactacacca gccccacctt
tggcggcgga 300acaaagctgg aaatcaagcg ggcc 32470222PRTHomo sapiens
70Gln Leu Pro Pro Gly Lys Pro Glu Ile Phe Lys Cys Arg Ser Pro Asn1
5 10 15Lys Glu Thr Phe Thr Cys Trp Trp Arg Pro Gly Thr Asp Gly Gly
Leu 20 25 30Pro Thr Asn Tyr Ser Leu Thr Tyr His Arg Glu Gly Glu Thr
Leu Met 35 40 45His Glu Cys Pro Asp Tyr Ile Thr Gly Gly Pro Asn Ser
Cys His Phe 50 55 60Gly Lys Gln Tyr Thr Ser Met Trp Arg Thr Tyr Ile
Met Met Val Asn65 70 75 80Ala Thr Asn Gln Met Gly Ser Ser Phe Ser
Asp Glu Leu Tyr Val Asp 85 90 95Val Thr Tyr Ile Val Gln Pro Asp Pro
Pro Leu Glu Leu Ala Val Glu 100 105 110Val Lys Gln Pro Glu Asp Arg
Lys Pro Tyr Leu Trp Ile Lys Trp Ser 115 120 125Pro Pro Thr Leu Ile
Asp Leu Lys Thr Gly Trp Phe Thr Leu Leu Tyr 130 135 140Glu Ile Arg
Leu Lys Pro Glu Lys Ala Ala Glu Trp Glu Ile His Phe145 150 155
160Ala Gly Gln Gln Thr Glu Phe Lys Ile Leu Ser Leu His Pro Gly Gln
165 170 175Lys Tyr Leu Val Gln Val Arg Cys Lys Pro Asp His Gly Tyr
Trp Ser 180 185 190Ala Trp Ser Pro Ala Thr Phe Ile Gln Ile Pro Ser
Asp Phe Thr Met 195 200 205Asn Asp Ile Glu Gly Arg Met Asp His His
His His His His 210 215 22071666DNAHomo sapiens 71cagttacctc
ctggaaaacc tgagatcttt aaatgtcgtt ctcccaataa ggaaacattc 60acctgctggt
ggaggcctgg gacagatgga ggacttccta ccaattattc actgacttac
120cacagggaag gagagacact catgcatgaa tgtccagact acataaccgg
tggccccaac 180tcctgccact ttggcaagca gtacacctcc atgtggagga
catacatcat gatggtcaat 240gccactaacc agatgggaag cagtttctcg
gatgaacttt atgtggacgt gacttacata 300gttcagccag accctccttt
ggagctggct gtggaagtaa aacagccaga agacagaaaa 360ccctacctgt
ggattaaatg gtctccacct accctgattg acttaaaaac tggttggttc
420acgctcctgt atgaaattcg attaaaaccc gagaaagcag ctgagtggga
gatccatttt 480gctgggcagc aaacagagtt taagattctc agcctacatc
caggacagaa ataccttgtc 540caggttcgct gcaaaccaga ccatggatac
tggagtgcat ggagtccagc gaccttcatt 600cagataccta gtgacttcac
catgaatgat atcgagggcc gcatggacca ccaccaccac 660caccac
66672222PRTMus musculus 72Gln Ser Pro Pro Gly Lys Pro Glu Ile His
Lys Cys Arg Ser Pro Asp1 5 10 15Lys Glu Thr Phe Thr Cys Trp Trp Asn
Pro Gly Ser Asp Gly Gly Leu 20 25 30Pro Thr Asn Tyr Ser Leu Thr Tyr
Ser Lys Glu Gly Glu Lys Asn Thr 35 40 45Tyr Glu Cys Pro Asp Tyr Lys
Thr Ser Gly Pro Asn Ser Cys Phe Phe 50 55 60Ser Lys Gln Tyr Thr Ser
Ile Trp Lys Ile Tyr Ile Ile Thr Val Asn65 70 75 80Ala Thr Asn Glu
Met Gly Ser Ser Thr Ser Asp Pro Leu Tyr Val Asp 85 90 95Val Thr Tyr
Ile Val Glu Pro Glu Pro Pro Arg Asn Leu Thr Leu Glu 100 105 110Val
Lys Gln Leu Lys Asp Lys Lys Thr Tyr Leu Trp Val Lys Trp Leu 115 120
125Pro Pro Thr Ile Thr Asp Val Lys Thr Gly Trp Phe Thr Met Glu Tyr
130 135 140Glu Ile Arg Leu Lys Ser Glu Glu Ala Asp Glu Trp Glu Ile
His Phe145 150 155 160Thr Gly His Gln Thr Gln Phe Lys Val Phe Asp
Leu Tyr Pro Gly Gln 165 170 175Lys Tyr Leu Val Gln Thr Arg Cys Lys
Pro Asp His Gly Tyr Trp Ser 180 185 190Arg Trp Gly Gln Glu Lys Ser
Ile Glu Ile Pro Asn Asp Phe Thr Leu 195 200 205Lys Asp Ile Glu Gly
Arg Met Asp His His His His His His 210 215 22073666DNAMus musculus
73cagtcaccac ctggaaaacc tgaaatccac aaatgtcgtt cccctgacaa ggaaacattc
60acctgctggt ggaatcctgg gtcagatgga ggactcccca ccaattattc attgacatac
120agcaaagaag gagagaaaaa cacctatgaa tgtccagact acaaaaccag
tggccccaat 180tcctgtttct ttagcaagca gtacacttcc atatggaaaa
tatacatcat cacagtaaat 240gccacgaacg aaatgggaag cagtacctcg
gatccacttt atgtggatgt gacttacatt 300gttgaaccag agcctcctcg
gaacctgact ttagaagtga aacaactaaa agacaaaaaa 360acatatctgt
gggtaaaatg gttgccacct accataactg atgtaaaaac tggttggttt
420acaatggaat atgaaattcg attaaagtct gaagaagcag atgagtggga
gatccacttc 480acaggtcatc aaacacaatt taaggttttt gacttatatc
caggacaaaa gtatcttgtc 540cagactcgct gcaagccaga ccatggatac
tggagtagat ggggccagga gaaatctatt 600gaaataccaa atgacttcac
cttgaaagac atcgagggcc gcatggacca ccaccaccac 660caccac 666
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