U.S. patent application number 12/112587 was filed with the patent office on 2009-01-15 for antibodies to the human prolactin receptor.
This patent application is currently assigned to BIOGEN IDEC MA INC.. Invention is credited to Brian Elenbaas, Stephen E. Fawell, Matthew B. Jarpe, Steven D. Miklasz.
Application Number | 20090017044 12/112587 |
Document ID | / |
Family ID | 38776399 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017044 |
Kind Code |
A1 |
Elenbaas; Brian ; et
al. |
January 15, 2009 |
Antibodies to the Human Prolactin Receptor
Abstract
This invention provides antibodies to the prolactin receptor,
particularly the human prolactin receptor. Preferred antibodies are
capable of blocking prolactin binding to the prolactin receptor,
inhibiting signaling through the prolactin receptor, and/or
inhibiting proliferation of cancer cells induced by prolactin. Also
provided are nucleic acids encoding the antibodies, vectors and
host cells comprising the nucleic acids, and uses of the antibodies
and nucleic acids.
Inventors: |
Elenbaas; Brian; (Melrose,
MA) ; Jarpe; Matthew B.; (Quincy, MA) ;
Miklasz; Steven D.; (Upton, MA) ; Fawell; Stephen
E.; (Framingham, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
38776399 |
Appl. No.: |
12/112587 |
Filed: |
April 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11538635 |
Oct 4, 2006 |
7422899 |
|
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12112587 |
|
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60724054 |
Oct 5, 2005 |
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Current U.S.
Class: |
424/172.1 ;
424/130.1; 435/252.3; 435/320.1; 435/325; 435/346; 435/375;
435/69.6; 514/44R; 530/387.1; 530/387.3; 530/391.3; 536/23.53 |
Current CPC
Class: |
C07K 16/2869 20130101;
A61P 31/00 20180101; A61K 2039/505 20130101; C07K 2317/73 20130101;
C07K 2317/76 20130101 |
Class at
Publication: |
424/172.1 ;
435/346; 530/387.1; 530/387.3; 530/391.3; 424/130.1; 536/23.53;
514/44; 435/320.1; 435/252.3; 435/325; 435/69.6; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/06 20060101 C12N005/06; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12P 21/04 20060101
C12P021/04; A61P 31/00 20060101 A61P031/00; C12N 1/20 20060101
C12N001/20; A61K 31/7088 20060101 A61K031/7088; C07K 16/18 20060101
C07K016/18 |
Claims
1. A hybridoma deposited at the American Type Culture Collection
(ATCC) as PTA-6477, PTA-6478, PTA-6479, PTA-6480, or PTA-6481.
2. An isolated antibody produced by the hybridoma of claim 1.
3. An isolated antibody recognizing the same epitope as the
antibody of claim 2.
4. The antibody of claim 3 that is a humanized antibody, chimeric
antibody, single chain antibody, Fab fragment, Fab' fragment,
F(ab')2 fragment, or Fv fragment.
5. The antibody of claim 3 comprising a detectable label.
6. A pharmaceutical composition comprising the antibody of claim
2.
7. The pharmaceutical composition of claim 6 further comprising a
chemotherapeutic agent.
8. The pharmaceutical composition of claim 6 further comprising an
additional antibody.
9. The pharmaceutical composition of claim 8 wherein the additional
antibody recognizes the human prolactin receptor.
10. An isolated nucleic acid comprising a sequence encoding the
antibody of claim 2.
11. A pharmaceutical composition comprising the nucleic acid of
claim 10.
12. A vector comprising the nucleic acid of claim 10.
13. A host cell comprising the vector of claim 12.
14. The host cell of claim 13 that is bacterial or mammalian.
15. A method of producing an antibody, comprising culturing the
host cell of claim 13 and harvesting the antibody from the culture
of the host cell.
16. The method of claim 15 wherein the antibody is harvested from
the supernatant of the host cell.
17. A method of inhibiting signaling through a prolactin receptor
in a cell, comprising contacting the cell with an effective amount
of the antibody of claim 2.
18. The method of claim 17 wherein the cell is a breast cancer
cell.
19. The method of claim 17 wherein the cell is a colon cancer cell,
prostate cancer cell, uterine cancer cell, leukemia cell or kidney
cancer cell.
20. A method of inhibiting prolactin-induced proliferation of a
cell, comprising contacting the cell with the antibody of claim
2.
21. The method of claim 20 wherein the cell is selected from the
group consisting of breast cancer cells, colon cancer cells,
prostate cancer cells, uterine cancer cells, leukemia cells, and
kidney cancer cells.
22. A method of treating breast cancer in a mammal, comprising
administering an effective amount of the antibody of claim 2 to the
mammal.
23. The method of claim 22 further comprising administering an
additional antibody to the mammal.
24. The method of claim 23 wherein the additional antibody
recognizes the human prolactin receptor.
25. The method of claim 23 wherein the additional antibody is
Herceptin.RTM..
26. The method of claim 22 further comprising administering a
chemotherapeutic agent to the mammal.
27. The method of claim 22 wherein the mammal is human.
28. An isolated antibody that recognizes a human prolactin receptor
but not mouse prolactin receptor, wherein the antibody inhibits
signaling through the human prolactin receptor.
29. The antibody of claim 28 that inhibits prolactin-induced
proliferation of a cell.
30. The antibody of claim 29 wherein the cell is a breast cancer
cell.
31. The antibody of claim 28 that binds to the human prolactin
receptor with an affinity of 1 nM or less.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application No. 60/724,054, filed Oct. 5, 2005. The prior
application is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] This invention relates to antibodies, particularly
antibodies to the human prolactin receptor and uses thereof.
BACKGROUND
[0003] Prolactin (PRL) is a neuroendocrine pituitary hormone that
is essential for lactogenesis in mammals. The prolactin receptor
(PRLR) is a type I transmembrane protein that belongs to the
cytokine hematopoeitic receptor superfamily. Both PRL and PRLR play
essential roles in breast development and lactation during
pregnancy. PRL is produced primarily in the pituitary gland during
pregnancy, and interacts with PRLR molecules on mammary epithelial
cells in the breast to cause cell proliferation and differentiation
during pregnancy. In addition to its primary role in breast
development and lactation during pregnancy, it has been postulated
that this hormone/receptor pair has additional physiological roles,
including regulation of certain aspects of the immune system.
Indeed, PRL has been found to be expressed from extrapituitary
sources such as the placenta, the normal breast and certain immune
cells.
[0004] PRL and PRLR have a long-suspected role in breast cancer for
several reasons. First, PRL over-expression in mouse models causes
mammary tumorigenesis. Second, administration of human PRL to
several human breast cancer cells in vitro stimulates their
proliferation. Third, a large epidemiological study has shown that
there is a positive correlation in women between their circulating
serum PRL levels and risk for developing breast cancer (Hankinson
et al., J. Natl. Cancer Inst. 91(7):629-34 (1999)). Lastly, several
reports indicate that both PRL and PRLR are over-expressed in a
high percentage of breast cancer specimens.
[0005] At the molecular level, PRL exerts its effects on cells by
binding to the PRLR, leading to receptor dimerization and
intracellular signaling. PRLR dimerization leads to activation of
the receptor via phosphorylation of certain tyrosine residues in
the cytoplasmic tail of the receptor. This in turn leads to
signaling and activation of the JAK/STAT and Ras pathways.
Hyperactivation of Ras signaling is a common feature in a wide
variety of human cancers and is known to stimulate cell
proliferation and cell survival. Thus, there is strong molecular
evidence for a role of PRL signaling in breast cancer, and
inhibition of this signaling to MAPK and AKT would be expected to
have therapeutic benefit.
[0006] The discovery that PRLR and PRL expression is upregulated in
a high percentage of breast cancers and/or certain breast cancer
cell lines has led to the proposal that inhibition of PRL signaling
through its receptor in breast cancers could be therapeutically
useful to breast cancer patients. Accordingly, several therapeutic
strategies have been developed to inhibit signaling, such as the
development of PRL antagonists. One such antagonist is the mutant
PRL G129R, which has been shown to inhibit PRL signaling and cause
apoptosis in human breast cancer cell lines in vitro. G129R also
inhibits breast tumor growth in mouse xenograft models in vivo
(Chen et al., Int. J. Oncol. 20:813-818 (2002); Chen et al., Clin.
Cancer Res. 5:3583-3593 (1999)). Therefore, the PRL-PRLR signaling
pathway is a potential target for breast cancer therapy
development.
SUMMARY
[0007] The present invention provides antibodies that bind
specifically to the prolactin receptor (PRLR) with high affinities.
Preferred antibodies have the desired properties of being able to
inhibit prolactin (PRL) binding to PRLR, PRL-mediated signaling to
the JAK/STAT and RAS pathways, and PRL-mediated cell proliferation.
These antibodies are thus useful as therapeutics for cancers that
express PRLR, especially breast cancers.
[0008] Accordingly, one aspect of the present invention provides an
isolated antibody that recognizes a human prolactin receptor and
preferably does not bind the mouse prolactin receptor, wherein the
antibody inhibits signaling through the human prolactin receptor.
Preferably, the antibody inhibits prolactin-induced proliferation
of a cell, such as a breast cancer cell. The antibody binds to the
human prolactin receptor with an affinity that is preferably 10 nM
or less, more preferably 3 nM or less, even more preferably 1 nM or
less, and most preferably 0.3 nM or less.
[0009] In specific embodiments, the present invention provides a
hybridoma deposited at the American Type Culture Collection (ATCC)
as PTA-6477, PTA-6478, PTA-6479, PTA-6480, or PTA-6481, as well as
an antibody that is produced directly from any of these hybridomas.
Also provided are antibodies that recognize the same epitope as an
antibody directly produced by any of these hybridomas. Such
antibodies include, without being limited to, humanized antibodies,
chimeric antibodies, single chain antibodies, Fab fragments, Fab'
fragments, F(ab')2 fragments, and Fv fragments. The antibody of the
present invention may also be an immunoconjugate, such as an
antibody comprising a detectable label.
[0010] Another aspect of the present invention provides a
pharmaceutical composition comprising the antibody of the present
invention. The pharmaceutical composition may further comprise an
additional antibody and/or a chemotherapeutic agent. The additional
antibody may or may not recognize the prolactin receptor. For
example, the additional antibody may be Herceptin.RTM..
[0011] Yet another aspect of the present invention provides a
nucleic acid that comprises a sequence encoding the antibody of the
present invention, pharmaceutical compositions comprising the
nucleic acid, vectors comprising the nucleic acid, and host cells
comprising the vector. The vector may be any plasmid, including
expression vectors. The host cell may be any suitable host cell,
such as bacterial, yeast, insect, and mammalian cells. Also
provided is a method of producing an antibody comprising culturing
the host cell and harvesting the antibody from the culture of the
host cell. For example, the antibody may be harvested from the
culture supernatant.
[0012] Another aspect of the present invention provides a kit
comprising at least one antibody of the present invention. The kit
may also comprise an additional antibody, a means for detecting the
antibody of the invention, a means for administering the antibody,
and/or package instructions.
[0013] A further aspect of the present invention provides a method
of inhibiting signaling through a prolactin receptor in a cell,
comprising contacting the cell with an effective amount of the
antibody of the present invention. The cell is preferably a breast
cancer cell. Alternatively, the cell may be a colon cancer cell,
prostate cancer cell, uterine cancer cell, leukemia cell or kidney
cancer cell. Preferably, the cell is a cancer cell over-expressing
the prolactin receptor.
[0014] Another aspect of the present invention provides a method of
inhibiting prolactin-induced proliferation of a cell, comprising
contacting the cell with the antibody of the present invention.
Such inhibition may be measured by any method known in the art and
is preferably at least about 20%, more preferably at least about
40%, even more preferably at least about 60%, and most preferably
at least about 80%. The cell is preferably a breast cancer cell.
Alternatively, the cell may be a colon cancer cell, prostate cancer
cell, uterine cancer cell, leukemia cell or kidney cancer cell.
Preferably, the cell is a cancer cell over-expressing the prolactin
receptor.
[0015] Also provided is a method of treating breast cancer in a
mammal, comprising administering an effective amount of the
antibody of the present invention to the mammal. Optionally, an
effective amount of an additional antibody or a chemotherapeutic
agent may also be administered to the mammal. The additional
antibody may or may not recognize the prolactin receptor. For
example, the additional antibody may be Herceptin.RTM.. The mammal
is preferably a human.
[0016] Similarly provided are methods of treating a cancer that
expresses PRLR, comprising administering an effective amount of the
antibody of the present invention to the mammal. The methods are
useful for the treatment of, for example, colon cancer, prostate
cancer, uterine cancer, leukemia and kidney cancer. The cancer
preferably over-expresses PRLR.
[0017] Another aspect of the present invention provides a method
for diagnosing or staging a PRLR-expressing cancer using an
antibody of the present invention. The cancer preferably
over-expresses PRLR. Examples of the cancer include breast cancer,
colon cancer, prostate cancer, uterine cancer, leukemia and kidney
cancer.
[0018] Yet another aspect provides a method of removing a
PRLR-expressing cell from a population of mixed cells. The
population of mixed cells may be hematopoietic stem cells suitable
for transplantation.
DESCRIPTION OF DRAWINGS
[0019] FIG. 1: Amino acid sequence of the PRLR-Fc fusion protein
used to immunize mice and screen for antibodies.
[0020] The amino acid sequence in this figure indicates amino acids
1-448 of the PRLR-Fc fusion protein used to immunize mice and for
subsequent screening of hybridoma clones by ELISA. The amino
terminal (N-terminal) amino acids 1-214 (bold) constitute the PRLR
extracellular domain of the expected mature sequence after cleavage
of the signal sequence. The initial amino acid Q represents amino
acid 25 of the uncleaved PRLR protein. PRLR amino acids 1-214 are
followed by 7 amino acids (underlined) that encode the TEV protease
(Tobacco Etch Virus) cleavage site. This site is followed by 227
amino acids (italics) encoding the human IgG.sub.1 Fc domain.
[0021] FIG. 2: FACS analysis showing that PRLR mAbs recognize PRLR
on human breast cancer cell lines. A. FACS analysis of the 5 PRLR
mAbs on PRLR-positive T-47D human breast cancer cells shows that
the 5 antibodies are able to bind to T-47D cells, while the control
MOPC-21 mouse IgG.sub.1 control antibody does not. B. A similar
FACS analysis on the PRLR-positive MCF-7 human breast cancer cells
shows that the PRLR antibodies, but not the control MOPC-21
antibody, recognize PRLR on the surface of these cells. MFI
indicates mean fluorescence intensity of the secondary goat
anti-mouse PE antibody bound to the indicated primary antibodies.
FL2-H indicates fluorescence channel 2, which measures fluorescence
from the PE conjugated secondary antibody.
[0022] FIG. 3: FACS analysis demonstrates that PRLR mAbs are
specific for human PRLR.
[0023] The five PRLR mAbs were tested for their ability to
recognize human and mouse PRLR by determining their binding
activities to 4 different cell populations: A. 293T human kidney
cells transiently transfected with a human PRLR expression
construct; B. 293T cells transiently transfected with a mouse PRLR
expression construct; C. PRLR-positive MX-1 human breast cancer
cells, and D. PRLR-positive HC-11 mouse mammary epithelial cells.
MFI: mean fluorescence intensity.
[0024] FIG. 4: Signaling Assay for Inhibition for PRL
Signaling.
[0025] T-47D human breast cancer cells, treated by prolactin (Prl)
where indicated, were lysed and 40 ug total lysate was loaded onto
a 10% SDS-PAGE gel for immunoblot analysis. The blot was probed
with mAbs to phospho-STAT5A/B (P-STAT), phospho-p44/42 MAPK
(P-MAPK), phospho-AKT (P-AKT), and total AKT (AKT, as a loading
control).
[0026] FIG. 5: Inhibition of PRL-mediated signaling by PRLR mAbs.
Results of a .sup.3H-thymidine cell proliferation assay using T-47D
human breast cancer cells are shown.
DETAILED DESCRIPTION
[0027] The present invention provides antibodies that bind
specifically to PRLR with high affinities, and uses thereof.
Preferred antibodies have the desired properties of being able to
inhibit PRL binding to PRLR, PRL-mediated signaling to the JAK/STAT
and RAS pathways, and PRL-mediated cell proliferation. These
antibodies are thus useful as therapeutics for cancers that express
PRLR, especially breast cancers.
[0028] Prior to describing the invention in further detail, the
terms used in this application are defined as follows unless
otherwise indicated.
I. DEFINITIONS
[0029] Antibody or Immunoglobulin. In one embodiment, the term
"antibody" or "immunoglobulin" molecules encompasses immunospecific
fragments thereof, e.g., naturally occurring antibody or
immunoglobulin molecules or engineered antibody molecules or
fragments that bind antigen in a manner similar to antibody
molecules. The terms "antibody" and "immunoglobulin" are used
interchangeably herein. An antibody or immunoglobulin comprises at
least the variable domain of a heavy chain, and normally comprises
at least the variable domains of a heavy chain and a light chain.
Basic immunoglobulin structures in vertebrate systems are
relatively well understood. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988). The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, chimeric antibodies, humanized
antibodies, human antibodies, multispecific antibodies (e.g.,
bispecific antibodies), fusion antibodies, immunoconjugates, and
antibody fragments, so long as they exhibit the desired
specificity.
[0030] As will be discussed in more detail below, the term
"immunoglobulin" comprises five broad classes of polypeptides that
can be distinguished biochemically. All five classes are clearly
within the scope of the present invention, and the following
discussion will generally be directed to the IgG class of
immunoglobulin molecules. With regard to IgG, a standard
immunoglobulin molecule comprises two identical light chain
polypeptides of molecular weight approximately 23,000 Daltons, and
two identical heavy chain polypeptides of molecular weight
53,000-70,000. The four chains are typically joined by disulfide
bonds in a "Y" configuration wherein the light chains bracket the
heavy chains starting at the mouth of the "Y" and continuing
through the variable region.
[0031] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used functionally. In this regard, it will be
appreciated that the variable domains of both the light (V.sub.L)
and heavy (V.sub.H) chain portions determine antigen recognition
and specificity. Conversely, the constant domains of the light
chain (C.sub.L) and the heavy chain (C.sub.H1, C.sub.H2 or
C.sub.H3) confer important biological properties such as secretion,
transplacental mobility, Fc receptor binding, complement binding,
and the like. By convention the numbering of the constant region
domains increases as they become more distal from the antigen
binding site or amino-terminus of the antibody. The N-terminal
portion is a variable region and at the C-terminal portion is a
constant region; the C.sub.H3 and C.sub.L domains actually comprise
the carboxy-terminus of the heavy and light chain,
respectively.
[0032] Light chains are classified as either kappa or lambda
(.kappa., .lamda.). Each heavy chain class may be bound with either
a kappa or lambda light chain. In general, the light and heavy
chains are covalently bonded to each other, and the "tail" portions
of the two heavy chains are bonded to each other by covalent
disulfide linkages or non-covalent linkages when the
immunoglobulins are generated either by hybridomas, B cells or
genetically engineered host cells. In the heavy chain, the amino
acid sequences run from an N-terminus at the forked ends of the Y
configuration to the C-terminus at the bottom of each chain. Those
skilled in the art will appreciate that heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, (.gamma., .mu., .alpha.,
.delta., .epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, etc., are
well characterized and are known to confer functional
specialization. Modified versions of each of these classes and
isotypes are readily discernable to the skilled artisan in view of
the instant disclosure and, accordingly, are within the scope of
the instant invention.
[0033] As indicated above, the variable region allows the antibody
to selectively recognize and specifically bind epitopes on
antigens. That is, the V.sub.L domain and V.sub.H domain of an
antibody combine to form the variable region that defines a three
dimensional antigen binding site. This quaternary antibody
structure forms the antigen binding site present at the end of each
arm of the Y. More specifically, the antigen binding site is
defined by three complementarity determining regions (CDRs) on each
of the V.sub.H and V.sub.L chains. In some instances, e.g., certain
immunoglobulin molecules derived from camelid species or engineered
based on camelid immunoglobulins, a complete immunoglobulin
molecule may consist of heavy chains only, with no light chains.
See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).
[0034] In naturally occurring antibodies, the six "complementarity
determining regions" or "CDRs" present in each antigen binding
domain are short, non-contiguous sequences of amino acids that are
specifically positioned to form the antigen binding domain as the
antibody assumes its three dimensional configuration in an aqueous
environment. The remainder of the amino acids in the antigen
binding domains, referred to as "framework" regions, show less
inter-molecular variability. The framework regions largely adopt a
.beta.-sheet conformation and the CDRs form loops which connect,
and in some cases form part of, the .beta.-sheet structure. Thus,
framework regions act to form a scaffold that provides for
positioning the CDRs in correct orientation by inter-chain,
non-covalent interactions. The antigen binding domain formed by the
positioned CDRs defines a surface complementary to the epitope on
the immunoreactive antigen. This complementary surface promotes the
non-covalent binding of the antibody to its cognate epitope. The
amino acids comprising the CDRs and the framework regions,
respectively, can be readily identified for any given heavy or
light chain variable region by one of ordinary skill in the art,
since they have been precisely defined (see, "Sequences of Proteins
of Immunological Interest," Kabat, E., et al., U.S. Department of
Health and Human Services, (1983); and Chothia and Lesk, J. Mol.
Biol., 196:901-917 (1987), which are incorporated herein by
reference in their entireties).
[0035] In one embodiment, an antibody of the invention comprises at
least one heavy or light chain CDR of an antibody molecule. In
another embodiment, an antibody of the invention comprises at least
two CDRs from one or more antibody molecules. In another
embodiment, an antibody of the invention comprises at least three
CDRs from one or more antibody molecules. In another embodiment, an
antibody of the invention comprises at least four CDRs from one or
more antibody molecules. In another embodiment, an antibody of the
invention comprises at least five CDRs from one or more antibody
molecules. In another embodiment, an antibody of the invention
comprises at least six CDRs from one or more antibody molecules.
Exemplary antibody molecules comprising at least one CDR are known
in the art, and exemplary molecules that can be included in the
subject antibodies are described herein.
[0036] Antibodies or immunospecific fragments thereof for use in
the methods of the invention include, but are not limited to,
polyclonal, monoclonal, multispecific, human, humanized,
primatized, or chimeric antibodies, single chain antibodies,
epitope-binding fragments, e.g., Fab, Fab' and F(ab).sub.2, Fd,
Fvs, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv), fragments comprising either a V.sub.L
or V.sub.H domain, fragments produced by a Fab expression library,
and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to the antibodies disclosed herein). ScFv molecules are
known in the art and are described, e.g., in U.S. Pat. No.
5,892,019. Immunoglobulin or antibody molecules of the invention
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class
(e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and
IgA.sub.2) or subclass of immunoglobulin molecule.
[0037] Antibody fragments, including single-chain antibodies, may
comprise the variable region(s) alone or in combination with the
entirety or a portion of the following: hinge region, C.sub.H1,
C.sub.H2, and C.sub.H3 domains. Also included in the invention are
antigen-binding fragments that also comprise any combination of
variable region(s) with a hinge region, C.sub.H1, C.sub.H2, and
C.sub.H3 domains. Antibodies or immunospecific fragments thereof
for use in the diagnostic and therapeutic methods disclosed herein
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, donkey, rabbit, goat,
guinea pig, camel, llama, horse, or chicken antibodies. In another
embodiment, the variable region may be condricthoid in origin
(e.g., from sharks). As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human
immunoglobulin, and antibodies isolated from human immunoglobulin
libraries or from animals transgenic for one or more human
immunoglobulins and that do not express endogenous immunoglobulins,
as described infra and, for example in, U.S. Pat. No. 5,939,598 by
Kucherlapati et al.
[0038] As used herein, the term "heavy chain portion" includes
amino acid sequences derived from an immunoglobulin heavy chain. A
polypeptide comprising a heavy chain portion comprises at least one
of: a C.sub.H1 domain, a hinge (e.g., upper, middle, and/or lower
hinge region) domain, a C.sub.H2 domain, a C.sub.H3 domain, or a
variant or fragment thereof. For example, an antibody for use in
the invention may comprise a polypeptide chain comprising a
C.sub.H1 domain; a polypeptide chain comprising a C.sub.H1 domain,
at least a portion of a hinge domain, and a C.sub.H2 domain; a
polypeptide chain comprising a C.sub.H1 domain and a C.sub.H3
domain; a polypeptide chain comprising a C.sub.H1 domain, at least
a portion of a hinge domain, and a C.sub.H3 domain, or a
polypeptide chain comprising a C.sub.H1 domain, at least a portion
of a hinge domain, a C.sub.H2 domain, and a C.sub.H3 domain. In
another embodiment, a polypeptide of the invention comprises a
polypeptide chain comprising a C.sub.H3 domain. Further, an
antibody for use in the invention may lack at least a portion of a
C.sub.H2 domain (e.g., all or part of a C.sub.H2 domain). As set
forth above, it will be understood by one of ordinary skill in the
art that these domains (e.g., the heavy chain portions) may be
modified such that they vary in amino acid sequence from the
naturally occurring immunoglobulin molecule.
[0039] In the antibodies or immunospecific fragments thereof for
use in the diagnostic and treatment methods disclosed herein, the
heavy chain portions of one polypeptide chain of a multimer may be
identical to those on a second polypeptide chain of the multimer.
Alternatively, heavy chain portion-containing monomers for use in
the methods of the invention are not identical. For example, each
monomer may comprise a different target binding site, forming, for
example, a bispecific antibody.
[0040] The heavy chain portions of an antibody for use in the
diagnostic and treatment methods disclosed herein may be derived
from different immunoglobulin molecules. For example, a heavy chain
portion of a polypeptide may comprise a C.sub.H1 domain derived
from an IgG.sub.1 molecule and a hinge region derived from an
IgG.sub.3 molecule. In another example, a heavy chain portion can
comprise a hinge region derived, in part, from an IgG.sub.1
molecule and, in part, from an IgG.sub.3 molecule. In another
example, a heavy chain portion can comprise a chimeric hinge
derived, in part, from an IgG.sub.1 molecule and, in part, from an
IgG.sub.4 molecule.
[0041] As used herein, the term "light chain portion" includes
amino acid sequences derived from an immunoglobulin light chain.
Preferably, the light chain portion comprises at least one of a
V.sub.L or C.sub.L domain.
[0042] An isolated nucleic acid molecule encoding a non-natural
variant of a polypeptide derived from an immunoglobulin (e.g., an
immunoglobulin heavy chain portion or light chain portion) can be
created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of the
immunoglobulin such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations may be introduced by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
non-essential amino acid residues.
[0043] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, including basic side
chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a nonessential amino acid residue in an
immunoglobulin polypeptide is preferably replaced with another
amino acid residue from the same side chain family. In another
embodiment, a string of amino acids can be replaced with a
structurally similar string that differs in order and/or
composition of side chain family members.
[0044] Alternatively, in another embodiment, mutations may be
introduced randomly along all or part of the immunoglobulin coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be incorporated into antibodies for use in the
diagnostic and treatment methods disclosed herein and screened for
their ability to bind to the desired antigen, e.g., PRLR.
[0045] Antibodies or fragment thereof for use in the diagnostic and
therapeutic methods disclosed herein may be described or specified
in terms of the epitope(s) or portion(s) of a target polypeptide
that they recognize or specifically bind. The portion of an antigen
which specifically interacts with the antigen binding domain of an
antibody is an "epitope," or an "antigenic determinant." An antigen
may comprise a single epitope, but typically, an antigen comprises
at least two epitopes, and can include any number of epitopes,
depending on the size, conformation, and type of antigen. Antigens
are typically peptides or polypeptides, but can be any molecule or
compound or a combination of molecules or compounds. For example,
an organic compound, e.g., dinitrophenol or DNP, a nucleic acid, a
carbohydrate, or a mixture of any of these compounds either with or
without a peptide or polypeptide can be a suitable antigen. Thus,
for example, an "epitope" on a polypeptide may include a
carbohydrate side chain.
[0046] The minimum size of a peptide or polypeptide epitope is
thought to be about four to five amino acids. Peptide or
polypeptide epitopes preferably contain at least seven, more
preferably at least nine and most preferably between at least about
15 to about 30 amino acids. Since a CDR can recognize an antigenic
peptide or polypeptide in its tertiary form, the amino acids
comprising an epitope need not be contiguous, and in some cases,
may not even be on the same peptide chain. In the present
invention, peptide or polypeptide antigens preferably contain a
sequence of at least 4, at least 5, at least 6, at least 7, more
preferably at least 8, at least 9, at least 10, at least 15, at
least 20, at least 25, and, most preferably, between about 15 to
about 30 amino acids. Preferred peptides or polypeptides
comprising, or alternatively consisting of, antigenic epitopes are
at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or 100 amino acid residues in length.
[0047] By "specifically binds," it is generally meant that an
antibody binds to an epitope via its CDR, and that the binding
entails some complementarity between the CDR and the epitope.
According to this definition, an antibody is said to "specifically
bind" to an epitope when it binds to that epitope, via its CDR more
readily than it would bind to a random, unrelated epitope. The term
"specificity" is used herein to qualify the relative affinity by
which a certain antibody binds to a certain epitope. For example,
antibody "A" may be deemed to have a higher specificity for a given
epitope than antibody "B," or antibody "A" may be said to bind to
epitope "C" with a higher specificity than it has for related
epitope "D."
[0048] By "preferentially binds," it is meant that the antibody
specifically binds to an epitope more readily than it would bind to
a related, similar, homologous, or analogous epitope. Thus, an
antibody which "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though
such an antibody may cross-react with the related epitope.
[0049] By way of non-limiting example, an antibody may be
considered to bind a first epitope preferentially if it binds said
first epitope with a dissociation constant (KD) that is less than
the antibody's KD for the second epitope. In another non-limiting
example, an antibody may be considered to bind a first antigen
preferentially if it binds the first epitope with an affinity that
is at least one order of magnitude less than the antibody's KD for
the second epitope. In another non-limiting example, an antibody
may be considered to bind a first epitope preferentially if it
binds the first epitope with an affinity that is at least two
orders of magnitude less than the antibody's KD for the second
epitope.
[0050] In another non-limiting example, an antibody may be
considered to bind a first epitope preferentially if it binds the
first epitope with an off rate (k(off)) that is less than the
antibody's k(off) for the second epitope. In another non-limiting
example, an antibody may be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that
is at least one order of magnitude less than the antibody's k(off)
for the second epitope. In another non-limiting example, an
antibody may be considered to bind a first epitope preferentially
if it binds the first epitope with an affinity that is at least two
orders of magnitude less than the antibody's k(off) for the second
epitope.
[0051] An antibody for use in the diagnostic and treatment methods
disclosed herein may be said to bind a target polypeptide disclosed
herein or a fragment or variant thereof with an off rate (k(off))
of less than or equal to 5.times.10.sup.-2 sec.sup.-1, 10.sup.-2
sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1 or 10.sup.-3 sec.sup.-1.
More preferably, an antibody of the invention may be said to bind a
target polypeptide disclosed herein or a fragment or variant
thereof with an off rate (k(off)) less than or equal to
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1,
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1.
[0052] An antibody or fragment thereof for use in the diagnostic
and treatment methods disclosed herein may be said to bind a target
polypeptide disclosed herein or a fragment or variant thereof with
an on rate (k(on)) of greater than or equal to 10.sup.3 M.sup.-1
sec.sup.-1, 5.times.10.sup.3 M.sup.-1 sec.sup.-1, 10.sup.4 M.sup.-1
sec.sup.-1 or 5.times.10.sup.4 M.sup.-1 sec.sup.-1. More
preferably, an antibody of the invention may be said to bind a
target polypeptide disclosed herein or a fragment or variant
thereof with an on rate (k(on)) greater than or equal to 10.sup.5
M.sup.-1 sec.sup.1, 5.times.10.sup.5 M.sup.-1 sec.sup.-1, 10.sup.6
M.sup.-1 sec.sup.-1, or 5.times.10.sup.6 M.sup.-1 sec.sup.-1 or
10.sup.7 M.sup.-1 sec.sup.-1.
[0053] An antibody is said to competitively inhibit binding of a
reference antibody to a given epitope if it preferentially binds to
that epitope to the extent that it blocks, to some degree, binding
of the reference antibody to the epitope. Competitive inhibition
may be determined by any method known in the art, for example,
competition ELISA assays. An antibody may be said to competitively
inhibit binding of the reference antibody to a given epitope by at
least 90%, at least 80%, at least 70%, at least 60%, or at least
50%.
[0054] As used herein, the term "affinity" refers to a measure of
the strength of the binding of an individual epitope with the CDR
of an immunoglobulin molecule. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) at pages 27-28. As used herein, the term
"avidity" refers to the overall stability of the complex between a
population of immunoglobulins and an antigen, that is, the
functional combining strength of an immunoglobulin mixture with the
antigen. See, e.g., Harlow at pages 29-34. Avidity is related to
both the affinity of individual immunoglobulin molecules in the
population with specific epitopes, and also the valencies of the
immunoglobulins and the antigen. For example, the interaction
between a bivalent monoclonal antibody and an antigen with a highly
repeating epitope structure, such as a polymer, would be one of
high avidity.
[0055] Antibodies or immunospecific fragments thereof for use in
the diagnostic and therapeutic methods disclosed herein may also be
described or specified in terms of their cross-reactivity. As used
herein, the term "cross-reactivity" refers to the ability of an
antibody, specific for one antigen, to react with a second antigen;
a measure of relatedness between two different antigenic
substances. Thus, an antibody is cross reactive if it binds to an
epitope other than the one that induced its formation. The cross
reactive epitope generally contains many of the same complementary
structural features as the inducing epitope, and in some cases, may
actually fit better than the original.
[0056] For example, certain antibodies have some degree of
cross-reactivity, in that they bind related, but non-identical
epitopes, e.g., epitopes with at least 95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 65%, at
least 60%, at least 55%, and at least 50% identity (as calculated
using methods known in the art and described herein) to a reference
epitope. An antibody may be said to have little or no
cross-reactivity if it does not bind epitopes with less than 95%,
less than 90%, less than 85%, less than 80%, less than 75%, less
than 70%, less than 65%, less than 60%, less than 55%, and less
than 50% identity (as calculated using methods known in the art and
described herein) to a reference epitope. An antibody may be deemed
"highly specific" for a certain epitope, if it does not bind any
other analog, ortholog, or homolog of that epitope.
[0057] Antibodies or immunospecific fragments thereof for use in
the diagnostic and treatment methods disclosed herein may also be
described or specified in terms of their binding affinity to a
polypeptide of the invention. Preferred binding affinities include
those with a dissociation constant or Kd less than
5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M,
5.times.10.sup.-4 M, 10.sup.-4M, 5.times.10.sup.-5 M, 10.sup.-5 M,
5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M,
5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14M, 10.sup.-14
M, 5.times.10.sup.-15 M, or 10.sup.-15 M.
[0058] Antibodies or immunospecific fragments thereof for use in
the diagnostic and treatment methods disclosed herein may act as
agonists or antagonists of target polypeptides described herein.
For example, an antibody for use in the methods of the present
invention may function as an antagonist, blocking or inhibiting
PRLR activity.
[0059] "Sequence identity" is defined as the percentage of residues
in an amino acid or nucleic acid sequence that are identical after
aligning the sequence with a reference sequence and introducing
gaps, if necessary, to achieve maximal sequence identity. Methods
and computer programs for the alignment, such as BLAST, are well
known in the art.
[0060] As used herein, the term "binding site" or "binding domain"
refers to a region of a binding molecule, e.g., a binding
polypeptide (e.g., an antibody or fragment thereof), which is
responsible for specifically binding to a target molecule of
interest (e.g., an antigen, ligand, receptor, substrate or
inhibitor). Exemplary binding domains include antibody variable
domains, a receptor binding domain of a ligand, or a ligand binding
domain of a receptor or an enzymatic domain. A binding domain on an
antibody is referred to herein as an "antigen binding domain."
[0061] An antibody or immunogenic fragment for use in the
diagnostic and treatment methods disclosed herein may be
"multispecific," e.g., bispecific, trispecific or of greater
multispecificity, meaning that it recognizes and binds to two or
more different epitopes present on one or more different antigens
(e.g., proteins) at the same time. Thus, whether a binding molecule
is "monospecfic" or "multi specific," e.g., "bispecific," refers to
the number of different epitopes with which the binding molecule
reacts. Multi specific antibodies may be specific for different
epitopes of a target polypeptide described herein, or may be
specific for a target polypeptide as well as for a heterologous
epitope, such as a heterologous polypeptide or solid support
material.
[0062] As used herein the term "valency" refers to the number of
potential binding domains, e.g., antigen binding domains, present
in a binding molecule. Each binding domain specifically binds one
epitope. When a binding molecule comprises more than one binding
domain, each binding domain may specifically bind the same epitope
(for an antibody with two binding domains, termed "bivalent
monospecific") or to different epitopes (for an antibody with two
binding domains, termed "bivalent bispecific"). An antibody may
also be bispecific and bivalent for each specificity (termed
"bispecific tetravalent antibodies"). In another embodiment,
tetravalent minibodies or domain deleted antibodies can be
made.
[0063] Bispecific bivalent antibodies, and methods of making them,
are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;
5,821,333; and U.S. Application Publication Nos. 2003/020734 and
2002/0155537, the disclosures of all of which are incorporated by
reference herein. Bispecific tetravalent antibodies, and methods of
making them are described, for instance, in WO 02/096948 and WO
00/44788, the disclosures of both of which are incorporated by
reference herein. See generally, PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0064] As previously indicated, the subunit structures and three
dimensional configuration of the constant regions of the various
immunoglobulin classes are well known. As used herein, the term
"V.sub.H domain" includes the amino terminal variable domain of an
immunoglobulin heavy chain and the term "C.sub.H1 domain" includes
the first (most amino terminal) constant region domain of an
immunoglobulin heavy chain. The C.sub.H1 domain is adjacent to the
V.sub.H domain and is amino terminal to the hinge region of an
immunoglobulin heavy chain molecule.
[0065] As used herein the term "C.sub.H2 domain" includes the
portion of a heavy chain molecule that extends, e.g., from about
residue 244 to residue 360 of an antibody using conventional
numbering schemes (residues 244 to 360, Kabat numbering system; and
residues 231-340, EU numbering system; see Kabat E A et al. op.
cit.). The C.sub.H2 domain is unique in that it is not closely
paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two C.sub.H2 domains
of an intact native IgG molecule. It is also well documented that
the C.sub.H3 domain extends from the C.sub.H2 domain to the
C-terminal of the IgG molecule and comprises approximately 108
residues.
[0066] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the C.sub.H1 domain to the
C.sub.H2 domain. This hinge region comprises approximately 25
residues and is flexible, thus allowing the two N-terminal antigen
binding regions to move independently. Hinge regions can be
subdivided into three distinct domains: upper, middle, and lower
hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).
[0067] As used herein the term "disulfide bond" includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group. In most naturally occurring IgG
molecules, the C.sub.H1 and C.sub.L regions are linked by a
disulfide bond and the two heavy chains are linked by two disulfide
bonds at positions corresponding to 239 and 242 using the Kabat
numbering system (position 226 or 229, EU numbering system).
[0068] As used herein, the term "chimeric antibody" will be held to
mean any antibody wherein the immunoreactive region or site is
obtained or derived from a first species and the constant region
(which may be intact, partial or modified in accordance with the
instant invention) is obtained from a second species. In preferred
embodiments the target binding region or site will be from a
non-human source (e.g., mouse or primate) and the constant region
is human.
[0069] As used herein, the term "engineered antibody" refers to an
antibody in which the variable domain in either the heavy and light
chain or both is altered by at least partial replacement of one or
more CDRs from an antibody of known specificity and, if necessary,
by partial framework region replacement and sequence changing.
Although the CDRs may be derived from an antibody of the same class
or even subclass as the antibody from which the framework regions
are derived, it is envisaged that the CDRs will be derived from an
antibody of different class and preferably from an antibody from a
different species. An engineered antibody in which one or more
"donor" CDRs from a non-human antibody of known specificity is
grafted into a human heavy or light chain framework region is
referred to herein as a "humanized antibody." It may not be
necessary to replace all of the CDRs with the complete CDRs from
the donor variable region to transfer the antigen binding capacity
of one variable domain to another. Rather, it may only be necessary
to transfer those residues that are necessary to maintain the
activity of the target binding site. Given the explanations set
forth in, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and
6,180,370, it will be well within the competence of those skilled
in the art, either by carrying out routine experimentation or by
trial and error testing to obtain a functional engineered or
humanized antibody.
[0070] Alterations to the variable region notwithstanding, those
skilled in the art will appreciate that, in preferred embodiments,
the anti-PRLR antibodies of the instant invention may comprise
antibodies, or immunoreactive fragments thereof, in which at least
a fraction of one or more of the constant region domains has been
deleted or otherwise altered so as to provide desired biochemical
characteristics such as increased tumor localization or reduced
serum half-life when compared with an antibody of approximately the
same immunogenicity comprising a native or unaltered constant
region. In selected embodiments, the constant region of these type
of anti-PRLR antibodies will comprise a human constant region.
Modifications to the constant region compatible with the instant
invention comprise additions, deletions or substitutions of one or
more amino acids in one or more domains. That is, the anti-PRLR
antibodies disclosed herein may comprise alterations or
modifications to one or more of the three heavy chain constant
domains (C.sub.H1, C.sub.H2 or C.sub.H3) and/or to the light chain
constant domain (C.sub.L). In especially preferred embodiments the
modified antibodies will comprise domain deleted constructs or
variants wherein the entire C.sub.H2 domain has been removed
(.DELTA.C.sub.H2 constructs).
[0071] As used herein the term "properly folded polypeptide"
includes polypeptides (e.g., antibodies) in which all of the
functional domains comprising the polypeptide are distinctly
active. As used herein, the term "improperly folded polypeptide"
includes polypeptides in which at least one of the functional
domains of the polypeptide is not active. In one embodiment, a
properly folded polypeptide comprises polypeptide chains linked by
at least one disulfide bond and, conversely, an improperly folded
polypeptide comprises polypeptide chains not linked by at least one
disulfide bond.
[0072] As used herein the term "engineered" includes manipulation
of nucleic acid or polypeptide molecules by synthetic means (e.g.,
by recombinant techniques, in vitro peptide synthesis, by enzymatic
or chemical coupling of peptides or some combination of these
techniques).
[0073] As used herein, the terms "linked," "fused" or "fusion" are
used interchangeably. These terms refer to the joining together of
two more elements or components, by whatever means including
chemical conjugation or recombinant means. An "in-frame fusion"
refers to the joining of two or more open reading frames (ORFs) to
form a continuous longer ORF, in a manner that maintains the
correct reading frame of the original ORFs. Thus, the resulting
recombinant fusion protein is a single protein containing two ore
more segments that correspond to polypeptides encoded by the
original ORFs (which segments are not normally so joined in
nature.) Although the reading frame is thus made continuous
throughout the fused segments, the segments may be physically or
spatially separated by, for example, in-frame linker sequence.
[0074] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminal direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide.
[0075] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0076] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII.
[0077] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors.
[0078] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0079] An "antibody that inhibits the growth of tumor cells
expressing PRLR" or a "growth inhibitory antibody" is one which
binds to and results in measurable growth inhibition of cancer
cells expressing or over-expressing PRLR. Preferred growth
inhibitory anti-PRLR antibodies inhibit growth of PRLR-expressing
tumor cells (e.g., breast cancer cells) by at least 20%, more
preferably at least about 40%, even more preferably by at least
about 60%, and most preferably by at least about 80%, as compared
to control cells not treated with the antibody.
[0080] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by methods known in the art,
such as binding of annexin V, fragmentation of DNA, cell shrinkage,
dilation of endoplasmic reticulum, cell fragmentation, and/or
formation of membrane vesicles (called apoptotic bodies).
Preferably, the antibody which induces apoptosis is one which
results in about 2 to 50 fold, preferably about 5 to 50 fold, and
most preferably about 10 to 50 fold, induction of annexin binding
relative to untreated cell in an annexin binding assay.
[0081] A "PRLR-expressing cell" is a cell which expresses
endogenous or transfected PRLR on the cell surface. A
"PRLR-expressing cancer" is a cancer comprising cells that express
endogenous PRLR on the cell surface. A cancer which
"over-expresses" PRLR is one which has significantly higher levels
of PRLR at the cell surface compared to a noncancerous cell of the
same tissue type. Such over-expression may be caused by gene
amplification or by increased transcription or translation. PRLR
over-expression may be determined in a diagnostic or prognostic
assay by evaluating levels of the PRLR protein present on the
surface of a cell (e.g., via an immunohistochemistry assay; FACS
analysis). Alternatively, or additionally, one may measure levels
of PRLR-encoding nucleic acid or mRNA in the cell, e.g., via
fluorescent in situ hybridization (FISH), Southern blotting,
Northern blotting, or polymerase chain reaction (PCR) techniques,
such as real time quantitative PCR (RT-PCR). One may also study
PRLR over-expression by measuring shed antigen in a biological
fluid such as serum, e.g., using antibody-based assays. Aside from
the above assays, various in vivo assays are available to the
skilled practitioner. For example, one may expose cells within the
body of the patient to an antibody which is optionally labeled with
a detectable label, e.g., a radioactive isotope, and binding of the
antibody to cells in the patient can be evaluated, e.g., by
external scanning for radioactivity or by analyzing a biopsy taken
from a patient previously exposed to the antibody.
[0082] A "mammal" refers to any mammal, including humans, domestic
and farm animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably,
the mammal is human.
[0083] By "hyperproliferative disease or disorder" is meant all
neoplastic cell growth and proliferation, whether malignant or
benign, including all transformed cells and tissues and all
cancerous cells and tissues. Hyperproliferative diseases or
disorders include, but are not limited to, precancerous lesions,
abnormal cell growths, benign tumors, malignant tumors, and
"cancer."
[0084] Additional examples of hyperproliferative diseases,
disorders, and/or conditions include, but are not limited to
neoplasms, whether benign or malignant, located in the: prostate,
colon, abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and peripheral), lymphatic system, pelvic, skin, soft
tissue, spleen, thoracic, and urogenital tract.
[0085] As used herein, the terms "tumor" or "tumor tissue" refer to
an abnormal mass of tissue that results from excessive cell
division. A tumor or tumor tissue comprises "tumor cells" which are
neoplastic cells with abnormal growth properties and no useful
bodily function. Tumors, tumor tissue and tumor cells may be benign
or malignant. A tumor or tumor tissue may also comprise
"tumor-associated non-tumor cells", e.g., vascular cells which form
blood vessels to supply the tumor or tumor tissue. Non-tumor cells
may be induced to replicate and develop by tumor cells, for
example, the induction of angiogenesis in a tumor or tumor
tissue.
[0086] As used herein, the term "malignancy" refers to a non-benign
tumor or a cancer. As used herein, the term "cancer" connotes a
type of hyperproliferative disease which includes a malignancy
characterized by deregulated or uncontrolled cell growth. Examples
of cancer include, but are not limited to, carcinoma, lymphoma,
blastoma, sarcoma, and leukemia or lymphoid malignancies. More
particular examples of such cancers are noted below and include:
squamous cell cancer (e.g., epithelial squamous cell cancer), lung
cancer including small-cell lung cancer, non-small cell lung
cancer, adenocarcinoma of the lung and squamous carcinoma of the
lung, cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal cancer, endometrial cancer or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,
anal carcinoma, penile carcinoma, as well as head and neck cancer.
The term "cancer" includes primary malignant cells or tumors (e.g.,
those whose cells have not migrated to sites in the subject's body
other than the site of the original malignancy or tumor) and
secondary malignant cells or tumors (e.g., those arising from
metastasis, the migration of malignant cells or tumor cells to
secondary sites that are different from the site of the original
tumor).
[0087] Other examples of cancers or malignancies include, but are
not limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult
Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of
the Renal Pelvis and Ureter, Central Nervous System (Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood
Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia,
Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma,
Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood
Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood
Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial
Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,
Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer,
Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma,
Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic
Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer,
Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ
Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia,
Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease,
Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,
Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung
Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0088] The method of the present invention may be used to treat
premalignant conditions and to prevent progression to a neoplastic
or malignant state, including but not limited to those disorders
described above. Such uses are indicated in conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, Basic Pathology, 2d Ed., W.B. Saunders Co.,
Philadelphia, pp. 68-79 (1976).
[0089] In preferred embodiments, the method of the invention is
used to inhibit growth, progression, and/or metastasis of cancers,
in particular those listed above.
[0090] Additional hyperproliferative diseases, disorders, and/or
conditions include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0091] Hyperplasia is a form of controlled cell proliferation,
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or function. Hyperplastic
disorders which can be treated by the method of the invention
include, but are not limited to, angiofollicular mediastinal lymph
node hyperplasia, angiolymphoid hyperplasia with eosinophilia,
atypical melanocytic hyperplasia, basal cell hyperplasia, benign
giant lymph node hyperplasia, cementum hyperplasia, congenital
adrenal hyperplasia, congenital sebaceous hyperplasia, cystic
hyperplasia, cystic hyperplasia of the breast, denture hyperplasia,
ductal hyperplasia, endometrial hyperplasia, fibromuscular
hyperplasia, focal epithelial hyperplasia, gingival hyperplasia,
inflammatory fibrous hyperplasia, inflammatory papillary
hyperplasia, intravascular papillary endothelial hyperplasia,
nodular hyperplasia of prostate, nodular regenerative hyperplasia,
pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia,
and verrucous hyperplasia.
[0092] Metaplasia is a form of controlled cell growth in which one
type of adult or fully differentiated cell substitutes for another
type of adult cell. Metaplastic disorders which can be treated by
the method of the invention include, but are not limited to,
agnogenic myeloid metaplasia, apocrine metaplasia, atypical
metaplasia, autoparenchymatous metaplasia, connective tissue
metaplasia, epithelial metaplasia, intestinal metaplasia,
metaplastic anemia, metaplastic ossification, metaplastic polyps,
myeloid metaplasia, primary myeloid metaplasia, secondary myeloid
metaplasia, squamous metaplasia, squamous metaplasia of amnion, and
symptomatic myeloid metaplasia.
[0093] Dysplasia is frequently a forerunner of cancer, and is found
mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation.
Dysplastic disorders which can be treated by the method of the
invention include, but are not limited to, anhidrotic ectodermal
dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia, mammary dysplasia, mandibulofacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[0094] Additional pre-neoplastic disorders which can be treated by
the method of the invention include, but are not limited to, benign
dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue hypertrophy, intestinal polyps, colon polyps,
and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease,
Farmer's Skin, solar cheilitis, and solar keratosis.
[0095] As used herein, the terms "treat" or "treatment" refer to
both therapeutic treatment and prophylactic or preventative
measures, wherein the object is to prevent or slow down (lessen) an
undesired physiological change or disorder, such as the development
or spread of cancer. Beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0096] For example, a subject is successfully "treated" for a
PRLR-expressing cancer if, after receiving a therapeutic amount of
an anti-PRLR antibody according to the methods of the present
invention, the patient shows one or more of the following at an
observable and/or measurable level: reduction in the number of
cancer cells; reduction in the tumor size; inhibition or
elimination of cancer cell infiltration into peripheral organs,
including the spread of cancer into soft tissue and bone;
inhibition or elimination of tumor metastasis; inhibition of tumor
growth; reduction of one or more of the symptoms associated with
the specific cancer; and reduced morbidity and mortality.
[0097] The above parameters for assessing successful treatment and
improvement in the disease can be determined by routine procedures
familiar to a physician. For cancer therapy, efficacy can be
measured, for example, by assessing the time to disease progression
and/or determining the response rate. Metastasis can be determined
by staging tests, bone scan, and tests for calcium level and other
enzymes to determine spread to the bone. Chest X-rays and
measurement of liver enzyme levels by known methods are used to
look for metastasis to the lungs and liver, respectively.
[0098] An "effective amount" is an amount sufficient for an
intended use. The term "therapeutically effective amount" refers to
an amount of an antibody or a drug effective to treat a disease or
disorder in a subject.
[0099] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to a cell or mammal at the dosages and concentrations employed.
Often the physiologically acceptable carrier is an aqueous pH
buffered solution. Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid; peptides; proteins,
such as serum albumin, gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN.TM., polyethylene glycol
(PEG), and PLURONICS.TM..
[0100] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125, Y90,
Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32 and
radioactive isotopes of Lu), chemotherapeutic agents, enzymes and
fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0101] A "detectable label" as used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0102] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0103] The term "package instructions" is used to refer to
instructions customarily included in commercial packages of
therapeutic products, that contain information about the
indications, usage, dosage, administration, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0104] An "isolated nucleic acid" is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other genome DNA, proteins, or complexes such as ribosomes and
polymerases, which naturally accompany a native sequence. The term
embraces a nucleic acid sequence which has been removed from its
naturally occurring environment, and includes recombinant or cloned
DNA isolates, chemically synthesized nucleic acids, and nucleic
acids biologically synthesized by heterologous systems.
II. COMPOSITIONS
[0105] To prepare anti-PRLR antibodies, a fusion protein comprising
the extracellular domain of the human PRLR and the Fc domain of
immunoglobulin was used to generate monoclonal antibodies (Example
1). The antibodies were screened for their binding activities, and
five hybridomas were further analyzed. These five hybridomas are
designated 4P1E9.C7, 4P1B3.3E8, 4P1E5.2G5, 4P4F1.C8, and 4P4H6.G3,
also referred to in this application as C7, 3E8, 2G5, C8 and G3,
respectively.
[0106] These hybridomas produce monoclonal antibodies that have
high affinities for the human PRLR, at the 10.sup.-8 M, 10.sup.-9
M, and 10.sup.-10 M levels (Example 1). The antibodies are specific
for human PRLR and do not bind the mouse PRLR (Example 2). However,
the antibodies do not compete with one another for binding to the
human PRLR. Therefore, they each recognize a different epitope in
the human PRLR (Example 1).
[0107] With the exception of C7, the antibodies blocked PRL binding
to PRLR (Example 3). Furthermore, the antibodies that blocked
PRL-PRLR binding also inhibited phosphorylation of MAPK, AKT and
STAT that is induced by PRL (Example 4). Therefore, the antibodies
produced by 3E8, 2G5, C8 and G3 can be used to inhibit PRL-PRLR
signaling. The antibodies from 3E8, C8 and G3 also effectively
inhibited T-47D breast cancer cell proliferation (Example 5).
Although the 2G5 antibody was not effective against T-47D cell
proliferation in the studies described herein, it is contemplated
that it may be used to inhibit cell growth of other cancer cells,
particularly cells with a higher PRLR level than T-47D cells.
[0108] The hybridomas were deposited at the American Type Culture
Collection (ATCC) under the Budapest Treaty on Dec. 21, 2004. The
ATCC designations for the hybridomas are as follows:
TABLE-US-00001 TABLE 1 ATCC designations of the hybridomas
Hybridoma Abbreviated as ATCC designation 4P1E9.C7 C7 PTA-6477
4P1B3.3E8 3E8 PTA-6478 4P1E5.2G5 2G5 PTA-6479 4P4F1.C8 C8 PTA-6480
4P4H6.G3 G3 PTA-6481
[0109] Accordingly, the present invention provides isolated
antibodies that recognize the human PRLR and preferably do not
recognize the mouse PRLR. The antibodies bind to the human PRLR
with an affinity of less than 10 nM, more preferably less than 3
nM, even more preferably less than 1 nM, and most preferably less
than 0.3 nM. Particularly provided are antibodies capable of
blocking the binding between PRL and the human PRLR receptor,
inhibiting PRL signal transduction, and/or inhibiting cancer cell
proliferation/survival.
[0110] In specific embodiments, the present invention provides a
hybridoma deposited at the ATCC as PTA-6477, PTA-6478, PTA-6479,
PTA-6480, or PTA-6481. Also provided are isolated antibodies that
recognize the same epitope as any one of these hybridomas. The
antibody may be directly produced from the hybridomas, or it may be
an antibody that recognizes the same epitope because it comprises
at least part of the antigen-binding regions of an antibody
produced by the hybridomas. These antibodies, and the preparation
thereof, are discussed in further detail below.
[0111] A. Production of Various Anti-PRLR Antibodies
[0112] Exemplary techniques are described for the production of the
antibodies useful in the present invention. The PRLR antigen to be
used for production of antibodies may be, e.g., the full length
polypeptide or a portion thereof. Alternatively, cells expressing
PRLR at their cell surface (e.g., CHO or NIH-3T3 cells transformed
to over-express PRLR; breast or other PRLR-expressing tumor cell
line), or membranes prepared from such cells, can be used to
generate antibodies. The nucleotide and amino acid sequences of
human and murine PRLR are available in the art. PRLR can be
produced recombinantly in and isolated from, bacterial or
eukaryotic cells using standard recombinant DNA methodology. PRLR
can be expressed as a tagged (e.g., epitope tag) fusion protein or
other fusion protein to facilitate isolation and identification in
various assays. Antibodies or binding proteins that bind to various
tags and fusion sequences are available as elaborated below. Other
forms of PRLR useful for generating antibodies will be apparent to
those skilled in the art.
[0113] 1. Tags
[0114] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)). The FLAG-peptide (Hopp et al.,
BioTechnology, 6:1204-1210 (1988)) is recognized by an anti-FLAG M2
monoclonal antibody (Eastman Kodak Co., New Haven, Conn.).
Purification of a protein containing the FLAG peptide can be
performed by immunoaffinity chromatography using an affinity matrix
comprising the anti-FLAG M2 monoclonal antibody covalently attached
to agarose (Eastman Kodak Co., New Haven, Conn.). Other tag
polypeptides include the KT3 epitope peptide (Martin et al.,
Science, 255:192-194 (1992)); an .alpha.-tubulin epitope peptide
(Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)); and the
T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)).
[0115] 2. Polyclonal Antibodies
[0116] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sub.1N.dbd.C.dbd.NR, where R and R.sub.1 are different alkyl
groups.
[0117] Animals are typically immunized against the antigen,
immunogenic conjugates, or derivatives by combining, e.g., 100
.mu.g or 5 .mu.g of the protein or conjugate (for rabbits or mice,
respectively) with 3 volumes of Freund's complete adjuvant and
injecting the solution intradermally at multiple sites. One month
later, the animals are boosted with 1/5 to 1/10 the original amount
of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later,
the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Conjugates also can
be made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum are suitably used to enhance the
immune response.
[0118] 3. Monoclonal Antibodies
[0119] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (e.g., U.S. Pat. No.
4,816,567).
[0120] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce antibodies specifically binding to
the protein used for immunization. Alternatively, lymphocytes may
be immunized in vitro. After immunization, lymphocytes are isolated
and then fused with a myeloma cell line using a suitable fusing
agent, such as polyethylene glycol, to form a hybridoma cell.
[0121] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner).
Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors, and SP-2 and
derivatives, e.g., X63-Ag8-653 cells. Human myeloma and mouse-human
heteromycloma cell lines also have been described for the
production of human monoclonal antibodies.
[0122] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. For example, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA). The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis or the methods
described herein.
[0123] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. In addition, the hybridoma cells may be grown in
vivo as ascites tumors in an animal, e.g., by i.p. injection of the
cells into mice.
[0124] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0125] Monoclonal antibodies or antibody fragments can also be
isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0126] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine
sequences, or by fusing the immunoglobulin coding sequence with all
or part of the coding sequence for a non-immunoglobulin polypeptide
(heterologous polypeptide). The non-immunoglobulin polypeptide
sequences can substitute for the constant domains of an antibody,
or they are substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for an antigen and another antigen-combining site having
specificity for a different antigen.
[0127] 4. Humanized Antibodies
[0128] Methods for humanizing non-human antibodies have been
described in the art. A humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
Humanization can be essentially performed following the method of
Winter and co-workers (Jones et al., Nature, 321:522-525 (1986);
Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0129] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0130] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0131] Various forms of a humanized anti-PRLR antibody are
contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG.sub.1 antibody.
[0132] 5. Human Antibodies
[0133] As an alternative to humanization, human antibodies can be
generated (e.g., van Dijk et al., Curr Opin Chem. Biol. 5(4):368-74
(2001)). For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice can result in the production of human
antibodies upon antigen challenge.
[0134] Alternatively, phage display technology can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B-cell.
[0135] Human antibodies may also be generated by in vitro activated
B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0136] A human antibody with a desired specificity, such as one
that recognizes the same epitope as the anti-PRLR hybridomas
described herein, can be identified by competitive ELISA or other
methods known in the art.
[0137] 6. Antibody Fragments
[0138] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. For example, the
smaller size of the fragments allows for rapid clearance, and may
lead to improved access to solid tumors.
[0139] Traditionally, antibody fragments were derived via
proteolytic digestion of intact antibodies. Alternatively, these
fragments can be produced directly by recombinant host cells. Fab,
Fv and ScFv antibody fragments can all be expressed in and secreted
from E. coli, thus allowing the facile production of large amounts
of these fragments. Antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab).sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice
is a single chain Fv fragment (scFv). Fv and sFv are the only
species with intact combining sites that are devoid of constant
regions; thus, they are suitable for reduced nonspecific binding
during in vivo use. sFv fusion proteins may be constructed to yield
fusion of an effector protein at either the amino or the carboxy
terminus of an sFv. The antibody fragment may also be a "linear
antibody", e.g., as described in U.S. Pat. No. 5,641,870. Such
linear antibody fragments may be monospecific or bispecific.
[0140] 7. Bispecific Antibodies
[0141] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
PRLR protein. Other such antibodies may combine an PRLR binding
site with a binding site for another protein. Alternatively, an
anti-PRLR arm may be combined with an arm which binds to a
triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g., CD3), or Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16),
so as to focus and localize cellular defense mechanisms to the
PRLR-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express PRLR. These
antibodies possess a PRLR-binding arm and an arm which binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies).
[0142] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature 305:537-539 (1983)). In a different
approach, antibody variable domains with the desired binding
specificities are fused to immunoglobulin constant domain
sequences. DNAs encoding the immunoglobulin heavy chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host cell. This provides for greater flexibility in adjusting the
proportions of the three polypeptide fragments. It is, however,
possible to insert the coding sequences for two or all three
polypeptide chains into a single expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields.
[0143] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0144] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Heteroconjugate antibodies may be made using any convenient
cross-linking methods.
[0145] The "diabody" technology provides an alternative mechanism
for making bispecific antibody fragments. The fragments comprise a
V.sub.H connected to a V.sub.L by a linker which is too short to
allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
[0146] 8. Multivalent Antibodies
[0147] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies with three or more
antigen binding sites (e.g., tetravalent antibodies), which can be
readily produced by recombinant expression of nucleic acid encoding
the polypeptide chains of the antibody. The multivalent antibody
can comprise a dimerization domain and three or more antigen
binding sites. The preferred dimerization domain comprises (or
consists of) an Fc region or a hinge region. The preferred
multivalent antibody herein comprises (or consists of) three to
about eight, but preferably four, antigen binding sites. The
multivalent antibody comprises at least one polypeptide chain (and
preferably two polypeptide chains), wherein the polypeptide
chain(s) comprise two or more variable domains. For instance, the
polypeptide chain(s) may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc,
wherein VD1 is a first variable domain, VD2 is a second variable
domain, Fc is one polypeptide chain of an Fc region, X1 and X2
represent an amino acid or peptide spacer, and n is 0 or 1.
[0148] 9. Other Amino Acid Sequence Modifications
[0149] Amino acid sequence modification(s) of the anti-PRLR
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the anti-PRLR antibody are prepared by introducing appropriate
nucleotide changes into the anti-PRLR antibody nucleic acid, or by
peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the anti-PRLR antibody.
Any combination of deletion, insertion, and substitution is made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the anti-PRLR antibody,
such as changing the number or position of glycosylation sites.
[0150] A useful method for identification of certain residues or
regions of the anti-PRLR antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis". In this
method, a residue or group of target residues are identified (e.g.,
charged residues such as arg, asp, his, lys, and glu) and replaced
by a neutral or negatively charged amino acid (most preferably
alanine or polyalanine) to affect the interaction of the amino
acids with PRLR antigen. Those amino acid locations demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at, or for, the sites of
substitution. For example, to analyze the performance of a mutation
at a given site, ala scanning or random mutagenesis is conducted at
the target codon or region and the expressed anti-PRLR antibody
variants are screened for the desired activity.
[0151] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-PRLR antibody with
an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the anti-PRLR
antibody molecule include the fusion to the N- or C-terminus of the
anti-PRLR antibody of an enzyme (e.g., for ADEPT) or a polypeptide
which increases the serum half-life of the antibody.
[0152] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-PRLR antibody molecule replaced by a different residue. The
sites of greatest interest for substitutional mutagenesis include
the hypervariable regions, but FR alterations are also
contemplated.
[0153] Any cysteine residue not involved in maintaining the proper
conformation of the anti-PRLR antibody also may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
[0154] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity). In order to identify candidate
hypervariable region sites for modification, alanine scanning
mutagenesis can be performed to identify hypervariable region
residues contributing significantly to antigen binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and human PRLR. Such contact
residues and neighboring residues are candidates for substitution
according to the techniques elaborated herein. Once such variants
are generated, the panel of variants is subjected to screening as
described herein and antibodies with superior properties in one or
more relevant assays may be selected for further development.
[0155] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0156] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0157] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0158] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
Homodimeric antibodies with enhanced anti-tumor activity may also
be prepared using heterobifunctional cross-linkers as described in
Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an
antibody can be engineered which has dual Fc regions and may
thereby have enhanced complement lysis and ADCC capabilities. See
Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
[0159] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in, e.g., U.S. Pat.
No. 5,739,277. A salvage receptor binding epitope is an epitope of
the Fc region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2,
IgG.sub.3, or IgG.sub.4) that is responsible for increasing the in
vivo serum half-life of the IgG molecule.
[0160] 10. Screening for Antibodies with the Desired Properties
[0161] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0162] The growth inhibitory effects of an anti-PRLR antibody of
the invention may be assessed by methods known in the art, e.g.,
using cells which express PRLR either endogenously or following
transfection with the PRLR gene. For example, the tumor cell lines
and PRLR-transfected cells provided in the Examples may be treated
with an anti-PRLR monoclonal antibody of the invention at various
concentrations for a few days (e.g., 2-7), and stained with crystal
violet or MTT. Another method of measuring proliferation would be
by comparing .sup.3H-thymidine uptake by the cells treated in the
presence or absence an anti-PRLR antibody of the invention.
Appropriate positive controls include treatment of a selected cell
line with a growth inhibitory antibody known to inhibit growth of
that cell line. Growth inhibition of tumor cells in vivo can be
determined in various ways, such as using a nude mouse bearing a
tumor graft. Preferably, the tumor cell is one that over-expresses
PRLR. Preferably, the anti-PRLR antibody inhibits cell
proliferation of a PRLR-expressing tumor cell in vitro or in vivo
by about 20-100% compared to the untreated tumor cell, more
preferably by about 40-100%, and even more preferably by about
60-100% or 80-100%, at an antibody concentration of about 0.5 to 30
.mu.g/ml. Growth inhibition can be measured at an antibody
concentration of about 0.5 to 30 .mu.g/ml or about 0.5 nM to 200 nM
in cell culture, where the growth inhibition is determined 1-10
days after exposure of the tumor cells to the antibody. The
antibody is growth inhibitory in vivo if administration of the
anti-PRLR antibody at about 1 .mu.g/kg to about 100 mg/kg body
weight results in reduction in tumor size or tumor cell
proliferation within about 5 days to 3 months from the first
administration of the antibody, preferably within about 5 to 30
days.
[0163] To select for antibodies which induce cell death, loss of
membrane integrity may be assessed as indicated by, e.g., propidium
iodide (PI), trypan blue or 7AAD uptake. A PI uptake assay can be
performed in the absence of complement and immune effector cells.
PRLR-expressing tumor cells are incubated with medium alone or
medium containing of the appropriate monoclonal antibody at, e.g.,
about 10 .mu./ml. The cells are incubated for a 3 day time period.
Following each treatment, cells are washed and clumps removed. The
cells then receive PI (10 .mu.g/ml) and are analyzed using a
FACSCAN.TM. flow cytometer and FACSCONVERT.TM. CellQuest software
(Becton Dickinson). Those antibodies which induce statistically
significant levels of cell death as determined by PI uptake may be
selected as cell death-inducing antibodies.
[0164] To screen for antibodies which bind to an epitope on PRLR
that is bound by an antibody of interest, a routine cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can
be performed. This assay can be used to determine if a test
antibody binds the same site or epitope as an anti-PRLR antibody of
the invention. Alternatively, or additionally, epitope mapping can
be performed by methods known in the art. For example, the antigen
sequence can be mutagenized such as by alanine scanning, to
identify contact residues. In a different method, peptides
corresponding to different regions of PRLR can be used in
competition assays with the test antibodies.
[0165] 11. Immunoconjugates
[0166] The invention also provides immunoconjugates comprising an
antibody conjugated to an anti-cancer agent such as a cytotoxic
agent or chemotherapeutic agents.
[0167] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[0168] Examples of other chemotherapeutic agents that can be
conjugated to the anti-PRLR antibodies of the invention include
BCNU, streptozoicin, vincristine and 5-fluorouracil, as well as
esperamicins.
[0169] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, .alpha.-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes.
[0170] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0171] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-PRLR antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, R.sup.188, Sm.sup.153, Bi.sup.212,
P.sup.32, Pb.sup.212 and radioactive isotopes of Lu. When the
conjugate is used for diagnosis, it may comprise a radioactive atom
for scintigraphic studies, for example Tc.sup.99m or I.sup.123, or
a spin label for nuclear magnetic resonance (NMR) imaging (also
known as magnetic resonance imaging, MRI), such as iodine.sup.123,
iodine.sup.131, indium.sup.111, fluorine.sup.19, carbon.sup.13,
nitrogen.sup.15, oxygen.sup.17, gadolinium, manganese or iron.
[0172] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine.sup.19 in place of hydrogen. Labels such as
Tc.sup.99m or I.sup.23, R.sup.186, R.sup.188 and In can be attached
via a cysteine residue in the peptide. Y.sup.90 can be attached via
a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem.
Biophys. Res. Commun. 80:49-57) can be used to incorporate
iodine.sup.123.
[0173] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody. The linker may
be a cleavable linker, facilitating release of the cytotoxic drug
in the cell.
[0174] Alternatively, a fusion protein comprising the anti-PRLR
antibody and cytotoxic agent may be made, e.g., by recombinant
techniques or peptide synthesis. The length of DNA may comprise
respective regions encoding the two portions of the conjugate
either adjacent one another or separated by a region encoding a
linker peptide which does not destroy the desired properties of the
conjugate.
[0175] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such as streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0176] 12. Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0177] The antibodies of the present invention may also be used in
ADET by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug to an active anti-cancer drug.
[0178] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs.
[0179] The enzymes can be covalently bound to the anti-PRLR
antibodies by techniques well known in the art such as the use of
the heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antibody of the invention linked to at least a
functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the
art.
[0180] 13. Other Antibody Modifications
[0181] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions.
[0182] The anti-PRLR antibodies disclosed herein may also be
formulated as immunoliposomes. The components of the liposome are
commonly arranged in a bilayer formation, similar to the lipid
arrangement of biological membranes. Liposomes containing the
antibody can be prepared by methods known in the art.
[0183] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired a
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes via a disulfide interchange
reaction. A chemotherapeutic agent can be optionally contained
within the liposome.
[0184] B. Nucleic Acids, Vectors and Host Cells
[0185] The invention also provides isolated nucleic acids encoding
the anti-PRLR antibodies of the present invention, vectors and host
cells comprising the nucleic acids, and methods for producing the
antibodies using the nucleic acids.
[0186] DNA encoding the monoclonal antibodies of the present
invention, in particular the C7, 3E8, 2G5, C8 and G3 antibodies,
can be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody). The DNA can then be inserted into a vector for
recombinant DNA manipulations (e.g., to make chimeric antibodies)
or protein expression. The vector components generally include, but
are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
[0187] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryoles for this purpose include eubacteria,
such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
lichenifonnis, Pseudomonas such as P. aeruginosa, and
Streptomyces.
[0188] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. After expression, the antibody is
isolated from the E. coli cell paste in a soluble fraction and can
be purified through, e.g., a protein A or G column depending on the
isotype.
[0189] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-PRLR antibody-encoding vectors. Saccharomyces cerevisiae,
or common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia;
Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa;
Schwanniomyces such as Schwanniomyces occidentalis; and filamentous
fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and
Aspergillus hosts such as A. nidulans and A. niger.
[0190] Suitable host cells for the expression of glycosylated
anti-PRLR antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopicius (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0191] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0192] Examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4);
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TR1 cells; MRC 5 cells; FS4
cells; and a human hepatoma line (Hep G2).
[0193] The antibody composition produced by the host cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma..sub.1, .gamma..sub.2, or .gamma..sub.4
heavy chains. Protein G is recommended for all mouse isotypes and
for human .gamma..sub.3. The matrix to which the affinity ligand is
attached is most often agarose, but other matrices are available.
Mechanically stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM. resin
(J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0194] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0195] C. Pharmaceutical Compositions
[0196] Pharmaceutical compositions comprising the antibodies of the
present invention can be prepared by mixing an antibody having the
desired degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (see, e.g., Gennaro, Remington:
The Science and Practice of Pharmacy, 20th Edition (Baltimore, Md.:
Lippincott Williams & Wilkins, 2000)) in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
acetate, Tris, phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); peptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; tonicifiers such as trehalose and sodium
chloride; sugars such as sucrose, mannitol, trehalose or sorbitol;
surfactant such as polysorbate; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM., or
polyethylene glycol (PEG). The antibody preferably comprises the
antibody at a concentration of between 5-200 mg/ml, preferably
between 10-100 mg/ml.
[0197] The pharmaceutical composition may also contain more than
one active compound for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, in addition to the
anti-PRLR antibody, it may be desirable to include an additional
antibody, e.g., a second anti-PRLR antibody which binds a different
epitope on PRLR, or an antibody to another target such as a growth
factor that affects the growth of the particular cancer. For
example, Herceptin.RTM. can be combined with the anti-PRLR antibody
to treat breast cancer, particularly estrogen-dependent breast
cancer. Alternatively or additionally, the composition may further
comprise a chemotherapeutic agent, cytotoxic agent, cytokine,
and/or anti-hormonal agent. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0198] The active ingredients may also be entrapped in
microcapsules (for example, hydroxymethylcellulose or
gelatin-microcapsules and polymethylmethacylate microcapsules), in
colloidal drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), or
in macroemulsions.
[0199] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides, copolymers of L-glutamic acid and .gamma.
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxyburyric acid.
[0200] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0201] D. Kits
[0202] Kits are also provided that are useful for various purposes,
e.g., for diagnosis, therapeutic uses, cell killing assays, or
purification or immunoprecipitation of PRLR from cells. In addition
to the antibodies of the present invention (or nucleic acids or
hybridoma thereof), the other components of the kits depend on the
purposes. For instance, for therapeutic uses, the kit may contain
additional therapeutic agents to be used in conjunction with the
antibodies. For diagnosis, the kits may comprise means to detect
antibody binding, such as secondary antibodies and reagents for
ELISA or other immunoassays. For isolation and purification of
PRLR, the kit can contain an anti-PRLR antibody coupled to beads
(e.g., sepharose beads). Kits can be provided which contain the
antibodies for detection and quantitation of PRLR in vitro, e.g.,
in an ELISA or a Western blot. The kit may comprise a container and
package instructions on or associated with the container. The
container holds a composition comprising at least one anti-PRLR
antibody of the invention. Additional containers may be included
that contain, e.g., diluents and buffers, or control antibodies.
The package instructions may provide a description of the
composition as well as instructions for the intended use.
III. METHODS
[0203] The anti-PRLR antibodies are useful for treating a
PRLR-expressing cancer or ameliorating one or more symptoms of the
cancer in a mammal. Such a cancer includes breast cancer, colon
cancer, prostate cancer, uterine cancer, kidney cancer, leukemia,
as well as metastatic cancers of any of the preceding. The cancer
preferably over-expresses PRLR. The therapeutic antibody may exert
anti-tumor affects by any one of a variety of mechanisms, such as
inhibiting cancer cell proliferation, inhibiting cell survival
signaling, inducing apoptosis, enabling the cells to be recognized
and killed by immune cells or complement, or enhancing their
sensitivity to chemotherapy.
[0204] The anti-PRLR antibodies of the invention also have various
non-therapeutic applications. The anti-PRLR antibodies of the
present invention can be useful for diagnosis and staging of
PRLR-expressing cancers (e.g., in radioimaging). The antibodies are
also useful for purification or immunoprecipitation of PRLR from
cells, as well as for detection and quantitation of PRLR in vitro,
e.g., in an ELISA or a Western blot. The antibodies can be used to
kill and eliminate PRLR-expressing cells from a population of mixed
cells as a step in the purification of other cells. In particular,
they can be used to purge cancer cells from hematopoietic stem
cells before transplantation of the stem cells, such as autologous
stem cell transplantation typically performed in conjunction with
high dose chemotherapy.
[0205] In addition, PRLR are expressed on cells of the immune
system (Clevenger et al., J Endocrinol 157, 187-197 (1998);
Pellegrini et al., Mol Endocrinol 6, 1023-1031 (1992)), including
circulating T cells. Consistent with this finding, PRLR has also
been reported to be expressed on the JURKAT human T cell leukemia
line. Anti-PRLR mAbs may thus be used as immune modulators.
[0206] For therapeutic applications, the anti-PRLR antibody can be
administered alone or in combination with other forms of
conventional therapy, either consecutively with, pre- or
post-conventional therapy. Currently, depending on the stage of the
cancer, breast cancer treatment involves one or a combination of
the following therapies: surgery, radiation therapy, hormonal
therapy, and chemotherapy. Anti-PRLR antibody therapy may be
especially desirable in elderly patients who do not tolerate the
toxicity and side effects of chemotherapy well, and in metastatic
disease where radiation therapy has limited use.
[0207] The anti-PRLR antibody can be administered with a
therapeutically effective dose of at least one chemotherapeutic
agent or a cocktail of chemotherapeutic agents. Cancer cells are
known to deregulate cell survival signaling pathways by increasing
the cell survival signals that enter a cell. In turn, enhanced cell
survival signaling is known to confer resistance to
chemotherapeutic drugs. Therefore, inhibition of cell survival
signaling pathway would be expected to enhance the effectiveness of
any standard-of-care chemotherapeutic drug for any cancer
indication. Therefore, it is contemplated that the anti-PRLR
antibodies that inhibit cell survival signaling pathway (e.g., AKT,
see Example 4) will increase the sensitivity of cancer cells to
chemotherapeutic agents.
[0208] Examples of chemotherapeutic agents include, without being
limited to, Adriamycin, Dactinomycin, Bleomycin, Vinblastine,
Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; aminoglutethimide; amsacrine; anastrozole;
anthramycin; asparaginase; asperlin; azacitidine; azetepa;
azotomycin; batimastat; benzodepa; bicalutamide; bisantrene
hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;
brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;
dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin;
dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin;
doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanolone propionate; duazomycin; edatrexate; eflomithine
hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;
epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole;
etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;
fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;
gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin
hydrochloride; ifosfamide; ilmofosine; interleukin 2 (including
recombinant interleukin 2, or rIL2), interferon (such as interferon
.alpha.-2a; interferon .alpha.-2b; interferon .alpha.-n1;
interferon .alpha.-n3; interferon .beta.-1a; interferon
.gamma.-1b); iproplatin; irinotecan hydrochloride; lanreotide
acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine;
peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone hydrochloride; plicamycin; plomestane; porfimer
sodium; porfiromycin; prednimustine; procarbazine hydrochloride;
puromycin; puromycin hydrochloride; pyrazofurin; riboprine;
rogletimide; safingol; safingol hydrochloride; semustine;
simtrazene; sparfosate sodium; sparsomycin; spirogermanium
hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur;
teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;
testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;
tirapazamine; toremifene citrate; trestolone acetate; triciribine
phosphate; trimetrexate; trimetrexate glucuronate; triptorelin;
tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;
verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;
vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin
hydrochloride.
[0209] Chemotherapeutic agents and biologics also include, but are
not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil;
abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;
aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;
amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole; andrographolide; angiogenesis inhibitors; antagonist
D; antagonist G; antarelix; anti-dorsalizing morphogenetic
protein-1; antiandrogen, prostatic carcinoma; antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate;
apoptosis gene modulators; apoptosis regulators; apurinic acid;
ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;
atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;
azasetron; azatoxin; azatyrosine; baccatin III derivatives;
balanol; batimastat; BCR/ABL antagonists; benzochlorins;
benzoylstaurosporine; beta lactam derivatives; .beta.-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide;
bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate; bropirimine; budotitane; buthionine
sulfoximine; calcipotriol; calphostin C; camptothecin derivatives;
canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorlns;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine;
docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;
duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;
eflomithine; elemene; emitefur; epirubicin; epristeride;
estramustine analogue; estrogen agonists; estrogen antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;
flezelastine; fluasterone; fludarabine; fluorodaunorunicin
hydrochloride; forfenimex; formestane; fostriecin; fotemustine;
gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase inhibitors; gemcitabine; glutathione inhibitors;
hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;
ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;
insulin-like growth factor-I receptor inhibitor; interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin;
4-ipomeanol; iroplact; irsogladine; isobengazole;
isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte a interferon; leuprolide+estrogen+progesterone;
leuprorelin; levamisole; liarozole; linear polyamine analogue;
lipophilic disaccharide peptide; lipophilic platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium
texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors; menogaril; merbarone; meterelin;
methioninase; metoclopramide; MIF inhibitor; mifepristone;
miltefosine; mirimostim; mismatched double stranded RNA;
mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin
fibroblast growth factor-saporin; mitoxantrone; mofarotene;
molgramostim; monoclonal antibody, human chorionic gonadotrophin;
mopidamol; multiple drug resistance gene inhibitor; multiple tumor
suppressor 1-based therapy; mustard anticancer agent; mycaperoxide
B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline;
N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;
nilutamide; nisamycin; nitric oxide modulators; nitroxide
antioxidant; nitrullyn; O.sub.6-benzylguanine; octreotide;
okicenone; oligonucleotides; onapristone; ondansetron; ondansetron;
oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain
antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate;
sodium phenylacetate; solverol; somatomedin binding protein;
sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0210] Examples of therapeutic antibodies that can be used include
but are not limited to HERCEPTIN.RTM. (Trastuzumab) (Genentech,
Calif.), which is a humanized anti-HER2 monoclonal antibody for the
treatment of patients with metastatic breast cancer; REOPRO.RTM.
(abciximab) (Centocor), which is an anti-glycoprotein IIb/IIIa
receptor on the platelets for the prevention of clot formation;
ZENAPAX.RTM. (daclizumab) (Roche Pharmaceuticals, Switzerland),
which is an immunosuppressive, humanized anti-CD25 monoclonal
antibody for the prevention of acute renal allograft rejection;
PANOREX.TM., which is a murine anti-17-IA cell surface antigen
IgG2a antibody (Glaxo Wellcome/Centocor); BEC2, which is a murine
anti-idiotype (GD3 epitope) IgG antibody (ImClone System);
IMC-C225, which is a chimeric anti-EGFR IgG antibody (ImClone
System); VITAXIN.TM., which is a humanized anti-.alpha.V.beta.3
integrin antibody (Applied Molecular Evolution/Medlmmune); Campath
1H/LDP-03, which is a humanized anti CD52 IgG.sub.1 antibody
(Leukosite); Smart M195, which is a humanized anti-CD33 IgG
antibody (Protein Design Lab/Kanebo); RITUXAN.TM., which is a
chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech,
Roche/Zettyaku); LYMPHOCIDE.TM., which is a humanized anti-CD22 IgG
antibody (Immunomedics); LYMPHOCIDE.TM. Y-90 (Immunomedics);
Lymphoscan (Tc-99m-labeled; radioimaging; Immunomedics); Nuvion
(against CD3; Protein Design Labs); CM3 is a humanized anti-ICAM3
antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody
(IDEC Pharm/Mitsubishi); ZEVALIN.TM. is a radiolabelled murine
anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized
anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4
antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody
(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG
(Protein Design Lab); 5G.1 is a humanized anti-complement factor 5
(C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-.alpha.
antibody (CAT/BASF); CDP870 is a humanized anti-TNF-.alpha.. Fab
fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1
antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human
anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CD20-sreptdavidin
(+biotin-yttrium 90; NeoRx); CDP571 is a humanized anti-TNF-.alpha.
IgG.sub.4 antibody (Celltech); LDP-02 is a humanized
anti-.alpha.4.beta.7 antibody (LeukoSite/Genentech); OrthoClone
OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech);
ANTOVA.TM. is a humanized anti-CD40L IgG antibody (Biogen);
ANTEGREN.TM. is a humanized anti-VLA-4 IgG antibody (Elan); and
CAT-152 is a human anti-TGF-.beta..sub.2 antibody (Cambridge Ab
Tech).
[0211] The Physicians' Desk Reference (PDR) discloses dosages of
chemotherapeutic agents that have been used in treatment of various
cancers. The dosing regimen and dosages of chemotherapeutic agents
that are therapeutically effective will depend on the particular
cancer being treated, the extent of the disease and other factors
familiar to the physician of skill in the art and can be determined
by the physician. As discussed above, it is contemplated that
anti-PRLR antibodies will sensitize cancer cells to
chemotherapeutic agents. Therefore, it is contemplated that a lower
dose of chemotherapeutic agents will be required when combined with
anti-PRLR antibodies.
[0212] In one particular embodiment, an immunoconjugate comprising
the anti-PRLR antibody conjugated with a cytotoxic agent is
administered to the patient. Preferably, the immunoconjugate bound
to the PRLR protein is internalized by the cell, resulting in
increased therapeutic efficacy of the immunoconjugate in killing
the cancer cell to which it binds. In a preferred embodiment, the
cytotoxic agent targets or interferes with the nucleic acid in the
cancer cell. Examples of such cytotoxic agents are described above
and include maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
[0213] The anti-PRLR antibodies or immunoconjugates are
administered to a subject in accord with known methods, such as
intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous or
subcutaneous administration of the antibody is preferred.
[0214] It may also be desirable to combine the anti-PRLR antibodies
with an antibody directed against another epitope of PRLR or
another target associated with the particular cancer. For example,
since C7, 3E8, 2G5, C8 and G3 recognize different epitopes in the
human PRLR, they can be used in combination. Herceptin.RTM., which
recognizes a different target in breast cancer therapy, can also be
used in combination with the anti-PRLR antibodies.
[0215] The antibody may be combined with an anti-hormonal compound;
e.g., an anti-estrogen compound such as tamoxifen; an
anti-progesterone such as onapristone; or an anti-androgen such as
flutamide, in dosages known for such molecules. Where the cancer to
be treated is estrogen independent cancer, the patient may
previously have been subjected to anti-estrogen therapy and, after
the cancer becomes estrogen independent, the anti-PRLR antibody
(and optionally other agents as described herein) may be
administered to the patient.
[0216] For the prevention or treatment of disease, the dosage and
mode of administration will be chosen by the physician according to
known criteria. The appropriate dosage of antibody will depend on
the type of disease to be treated, as defined above, the severity
and course of the disease, whether the antibody is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, and the discretion
of the attending physician. The antibody is suitably administered
to the patient at one time or over a series of treatments.
Preferably, the antibody is administered by intravenous infusion or
by subcutaneous injections. Depending on the type and severity of
the disease, about 1 .mu.g/kg to about 50 mg/kg body weight (e.g.,
about 0.1-15 mg/kg/dose) of antibody can be an initial candidate
dosage for administration to the patient, whether, for example, by
one or more separate administrations, or by continuous infusion.
For repeated administrations over several days or longer, depending
on the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. The progress of this
therapy can be readily monitored by conventional methods and assays
and based on criteria known to the physician or other persons of
skill in the art.
[0217] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy as a way to deliver the antibody. There
are two major approaches to getting the nucleic acid (optionally
contained in a vector) into the patient's cells; in vivo and ex
vivo. For in vivo delivery the nucleic acid is injected directly
into the patient, usually at the site where the antibody is
required. For ex vivo treatment, the patient's cells are removed,
the nucleic acid is introduced into these isolated cells and the
modified cells are administered to the patient either directly or,
for example, encapsulated within porous membranes which are
implanted into the patient. There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retroviral vector.
[0218] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex 1 virus, or adeno-associated virus) and
lipid-based systems (e.g., DOTMA, DOPE and DC-Chol).
[0219] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of the present invention.
EXAMPLES
[0220] In the examples below, the following abbreviations have the
following meanings. Abbreviations not defined have their generally
accepted meanings. [0221] .degree. C.=degree Celsius [0222] hr=hour
[0223] min=minute [0224] sec=second [0225] .mu.M=micromolar [0226]
mM=millimolar [0227] M=molar [0228] ml=milliliter [0229]
.mu.l=microliter [0230] mg=milligram [0231] .mu.g=microgram [0232]
PBS=phosphate buffered saline [0233] PE=phycoerythrin [0234]
PRL=prolactin [0235] PRLR prolactin receptor
Materials and Methods
Expression and Purification of PRLR-Fc Fusion Protein for
Immunization and Screening:
[0236] An expression plasmid was constructed to express a PRLR-Fc
fusion protein (see FIG. 1). The fusion protein contains the
extracellular portion of the human prolactin receptor at the
N-terminus, followed by a tev (Tobacco Etch Virus) cleavage site,
fused to the human IgG.sub.1 Fc domain. This PRLR-Fc fusion protein
was expressed in HEK.EBNA cells according to standard protocols,
and purified from the supernatant of the transfected cells by
standard protein-A affinity column purification (Harlow and Lane,
Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory,
1988). Briefly, the supernatant was passed through a protein A
affinity column and eluted in a low pH buffer (100 mM sodium
phosphate, pH 2.8), and dialyzed in PBS. The protein was
characterized by SDS-PAGE, N-terminal sequencing and mass
spectroscopy analysis. These methods revealed a single protein
product of the expected size and mass with >95% purity. This
PRLR-Fc fusion protein was also used for the ELISA assay.
[0237] The N-terminal PRLR extracellular domain was cleaved for
immunization in mice, by cleaving the PRLR-Fc fusion protein with
the TEV (Tobacco Etch Virus) protease according to a standard
protocol (Haspel et al., Biotechniques 30, 60-61, 64-66
(2001)).
Antibody Purification:
[0238] Monoclonal antibodies were purified from hybridoma
supernatants by standard protein A affinity chromatography (Harlow
and Lane, Antibodies, A Laboratory Manual. Cold Spring Harbor
Laboratory, 1988) for use in ELISA, FACS and cell-based signaling
and proliferation assays.
ELISA Assay to Identify Antibodies that Bind PRLR:
[0239] Antibodies were tested for binding to recombinant prolactin
receptor (PRLR-Fc fusion protein, FIG. 1) using a standard ELISA
assay. Briefly, the PRLR-Fc fusion protein was coated onto 96-well
plates at 5 .mu.g/ml in PBS overnight. The plates were rinsed and
incubated with candidate antibodies in PBS containing 0.05%
Tween-20 and 1% BSA for one hour at room temperature. The plates
were washed, and the amount of bound antibodies was determined with
goat anti-mouse IgG HRP conjugate.
FACS Assay to Show Antibody Binding and Specificity:
[0240] Following isolation of hybridoma clones that recognized
PRLR-Fc fusion protein by the ELISA assay, purified antibodies were
tested for their ability to recognize PRLR protein on the surface
of various cell lines by FACS. Briefly, cells were grown under
standard tissue culture conditions. For the transient expression in
293T cells, the expression constructs were transiently transfected
into the cells using Fugene (Roche) and analyzed 36 hr
post-transfection.
[0241] The cells were harvested by washing in PBS and incubating in
PBS plus 5 mM EDTA for minutes. Cells are resuspended in FACS
buffer (0.1% BSA in PBS) on ice, counted, and 1.times.10.sup.6
cells were placed in each well of a 96-well plate. The cells were
spun and resuspended in 150 ul FACS buffer containing 5 ug/ml
antibody. For all experiments, the MOPC-21 IgG.sub.1 control
antibody (BD Biosciences) was used as a negative control. After a
20-minute incubation on ice, cells were spun and resuspended in
FACS buffer containing the secondary antibody, goat anti-mouse PE
(Jackson ImmunoResearch), and incubated for 20 minutes. The cells
were then washed twice in FACS buffer, fixed in 1%
paraformaldehyde, and analyzed by FACS.
Binding Studies Using BIAcore Chips:
[0242] A BIAcore 2000.TM. biosensor system (BIAcore, Inc.) was used
to study the binding of various mAbs to immobilized PRLR-Fc, and
their effects on PRL binding to the same receptor. All experiments
were performed at 25.degree. C. with a 10 .mu.l/minute flow rate,
using HBS buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% P20
surfactant, at pH 7.4). The same solution was used both as running
buffer and as sample diluent.
[0243] The CM5 chip (BIAcore, Inc.) surface was first activated
with
N-hydroxysuccinimide/N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide
hydrochloride (BIAcore, Inc.). PRLR-Fc, diluted to 30 .mu.g/ml in
10 mM acetic acid (pH 5), was then injected. The un-reacted groups
of the chip's dextran matrix were then blocked once with 30 .mu.l
and again with 15 .mu.l of ethanolamine-HCl (pH 8.5). The chip was
regenerated with a 20 .mu.l injection of 1 mM formic acid, repeated
five times to establish a reproducible and stable baseline.
[0244] For the experiment, anti-PRLR mAbs and PRL were diluted to
30 .mu.g/ml in diluent buffer. For each run, 100 .mu.l of one of
the anti-PRLR-Fc mAbs was injected over the surface of the chip
followed by a 100 .mu.l injection of PRL over the
PRLR-Fc/anti-PRLR-Fc mAb complex. Immediately after each injection,
the chip was washed with 300 .mu.l of the diluent buffer. The
surface was regenerated between experiments by injecting 20 .mu.l
of 1 mM formic acid, followed by a 15 .mu.l injection of 1 mM
formic acid and then a 10 .mu.l injection of 1 mM formic acid.
After regeneration, the chip was equilibrated with the diluent
buffer.
FACS Assay to Determine the Functional Affinities of PRLR
Antibodies:
[0245] BT474 human breast cancer cells were grown in RPMI plus 10%
FBS. The cells were detached from culture flasks with PBS
containing 20 mM EDTA at room temperature for 5 minutes. The
resulting suspension was centrifuged, the cells resuspended in PBS,
and the cell suspension was added to antibodies at various
concentrations in a 96-well polypropylene plate. After a one-hour
incubation at 4.degree. C., the cells were washed three times in
PBS and incubated with a 1:500 dilution of anti-mouse IgG
phycoerythrin for one hour at 4.degree. C. The cells were then
washed once and analyzed by flow cytometry. The mean fluorescence
intensity (MFI) was plotted against antibody concentration, and the
resulting curve was fit using a four-parameter equation using
DeltaGraph software. If no saturation was reached in the binding
experiments, an approximate value for the affinity is determined by
estimating maximum binding based on the maximum MFI of the other
antibodies.
Identification of Epitope Groups for the Antibodies:
[0246] The antibodies were sorted into epitope groups by
competition ELISA. All antibodies were conjugated with biotin
(using a biotin XX linker kit from Molecular Probes) and tested for
binding to purified PRLR-Fc fusion protein. Each antibody was then
tested against another, unconjugated antibody to allow competition
for the fusion protein. All antibodies blocked their own
corresponding conjugated forms, but none of the antibodies blocked
the others.
Cell Signaling Assay:
[0247] T-47D cells were plated onto 60 mm tissue culture dishes in
media containing 10% FBS at .about.70% confluency
(.about.0.8.times.10.sup.6 cells/plate). The cells were allowed to
adhere for 4-6 hr before switching the media to identical media
that lack serum, and incubated overnight. The following day PRL
ligand (R&D Systems) was added at a concentration of 0.1 ug/ml
and incubated for 15 min. A Stop Buffer (PBS containing 0.4 mM
NaOrthovanadate) was then added, and the cells were scraped and
harvested into test tubes on ice. Cells were then spun briefly in a
centrifuge and the supernatant was removed. 40 ul of a Lysis Buffer
(10 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 10% glycerol, 0.5%
Triton X-100, Roche complete protease inhibitor cocktail, 50 mM
NaF, 30 mM Na Phosphate, 1 mM NaOrthovanadate) was then added to
the cell pellet. 50 ug total cell lysate was analyzed by SDS-PAGE
and immunoblotting was performed with the following antibodies:
Phosho STAT5 A/B Y694/Y699 (Upstate), Phospho AKT and total AKT
(Cell Signaling PhosphoPlus AKT Ser 473 Kit), and Phospho MAPK
(PhosphoPlus p44/42 MAPK Thr202/Tyr204 Kit).
Cell Proliferation Assay:
[0248] T-47D human breast cancer cells (ATCC) were plated in
96-well plates in a culture medium plus 10% FBS on Day 1 at 10,000
cells/well. This medium was changed to a medium containing 2%
charcoal stripped serum for an overnight incubation. On Day 2,
cells were treated with 5 ug/ml PRLR antibodies or the MOPC control
antibody (BD Biosciences) for 2 hours, followed by 0.1 ug/ml PRL
ligand (R&D Systems). On Day 3, cells were again treated with
the antibody and the ligand at the same time. Cell proliferation
was measured by H.sup.3-thymidine incorporation on Day 4.
Pharmacokinetic (PK) Analysis of Antibodies:
[0249] PK analysis was performed by injecting 1 mg/kg each antibody
intraperitoneally (i.p.) into female athymic nude mice. Blood
samples were taken at the following time points following injection
(3 mice per time point): 15 min, 30 min, 1 hr, 2 hr, 6 hr, 24 hr,
48 hr, 96 hr, 168 hr, 216 hr, 264 hr, and 336 hr.
Example 1
Production and Affinities of PRLR Specific Antibodies
[0250] To generate monoclonal antibodies against the human PRLR,
the PRLR extracellular domain of the expected mature sequence
(amino acid residues 25-238 of the nascent PRLR protein) was fused
to the human IgG.sub.1 Fc domain. A 7-amino acid protease cleavage
site was inserted in between so that the TEV protease (Tobacco Etch
Virus) can be used to release the PRLR extracellular domain from
the fusion protein. The fusion protein (see FIG. 1) was expressed
and purified, and used to raise monoclonal antibodies as described
in Materials and Methods.
[0251] The resulting hybriodoma clones were screened for their
binding activities to the fusion protein by ELISA as described in
Materials and Methods. Five hybridomas were chosen for further
analyses. These mAbs produced by these hybridomas all recognize
human PRLR on the cell surface of intact cells as shown in FACS
analyses. Thus, PRLR-positive T-47D or MCF-7 human breast cancer
cells were used to bind each of these mAbs, followed by
fluorescence-labeled secondary antibodies that recognize the mAbs.
The cells were then analyzed by FACS. As shown in FIG. 2, the mAbs
were capable of binding both T-47D (FIG. 2A) and MCF-7 cells (FIG.
2B). In contrast, the control MOPC-21 antibody did not bind either
T-47D or MCF-7 cells.
[0252] These five hybridomas have high binding affinities for human
PRLR, with affinities around the nM level. When the antibodies were
allowed to compete with one another in pairs in order to determine
if they bind to the same epitope (see Materials and Methods), none
of the antibodies competed with another. Therefore, each of the
antibodies belongs to a different epitope group, arbitrarily
assigned as Group A, B, C, D, or E. These characteristics are shown
in Table 2 below. The "fusion partner" indicates the fusion partner
in the cell fusion step during hybridoma preparation.
TABLE-US-00002 TABLE 2 Affinities of the PRLR antibodies
Abbreviated Fusion Functional Clone name name partner Affinity (nM)
Epitope Group 4P1E9.C7 C7 SP2/0 2.52 A 4P1B3.3E8 3E8 SP2/0 0.1 B
4P1E5.2G5 2G5 SP2/0 0.173 C 4P4F1.C8 C8 FL653 ~13 D 4P4H6.G3 G3
FL653 ~13 E
Example 2
[0253] The PRLR Antibodies Specifically Recognize Human PRLR
[0254] The five PRLR mAbs were tested for their abilities to
recognize human and mouse PRLR by FACS analyses using 4 different
cell populations: A) 293T human kidney cells transiently
transfected with a human PRLR expression construct, B) 293T cells
transiently transfected with a mouse PRLR expression construct, C)
PRLR-positive MX-1 human breast cancer cells, and D) PRLR-positive
HC-11 mouse mammary epithelial cells.
[0255] The results (FIG. 3) show that, as expected, the PRLR mAbs
recognized the 293T cells transiently transfected with human PRLR
and the MX-1 cells. However, they were unable to recognize the 293T
cells transiently transfected with mouse PRLR, or the HC11 mouse
cells. Therefore, all five mAbs are specific for the human
PRLR.
Example 3
The Antibodies Inhibit PRL Binding to PRLR
[0256] To determine if the antibodies block the binding between PRL
and PRLR, the BIAcore biosensor system was used as described in
Materials and Methods. Briefly, the PRLR-Fc fusion protein was
immobilized to the BIAcore chip, and a test mAb was allowed to bind
to the immobilized PRLR. Subsequently, PRL was added to the chip.
Binding of the antibodies and PRL to the chip was continuously
monitored during the course of the experiments. The results
indicate that 3E8, 2G5, C8 and G3, but not C7, all blocked binding
of PRL to PRLR.
Example 4
The Antibodies Inhibit PRL Signaling
[0257] Upon binding to its ligand PRL, PRLR signals through the
JAK/STAT and Ras pathways to exert its various functions, including
milk production, cell proliferation, and cell survival. More
specifically, activation of JAK2 and STAT5 via phosphorylation
leads to increased transcription of the milk protein gene
.beta.-casein. Activation of the Ras pathways activates, in turn,
downstream effectors MAPK and AKT via phosphorylation of those
proteins. Activation of MAPK typically enhances cell proliferation,
and activation of AKT is known to enhance cell survival.
[0258] To determine if the five mAbs can inhibit PRL signaling,
T-47D cells were treated with PRL and candidate mAbs as described
in Materials and Methods. Cell lysates were then prepared from the
treated cells and used in Western blot analyses with antibodies
that detect phosho STAT5, phospho AKT, and phospho MAPK,
respectively. Anti-total AKT antibodies were also employed as a
loading control. The results are shown in FIG. 4. With the
exception of C7, the mAbs significantly inhibited phosphorylation
of STAT, AKT and MAPK. The negative control antibody, MOPC, had no
effects. These results are consistent with those from EXAMPLE 3,
and indicate that the four inhibitory antibodies can be used to
inhibit phosphorylation of these effectors triggered by PRL.
Example 5
The Antibodies Inhibit Breast Cancer Cell Proliferation
[0259] To determine whether the mAbs inhibit breast cancer cell
proliferation, T-47D cells were incubated with PRL and candidate
antibodies in the presence of H.sup.3-thymidine, as described in
Materials and Methods. A few days later, the cells were harvested
and H.sup.3-thymidine incorporation measured. As shown in FIG. 5
and summarized in Table 3, 3E8, C8 and G3 significantly inhibited
T-47D cell proliferation. 2G5 was not as effective as 3E8, C8 and
G3 in inhibiting PRL signaling, and it did not significantly
inhibit T-47D cell proliferation. C7 and the control antibody MOPC,
which did not inhibit PRL signaling, had no effect on PRL-induced
T-47D cell proliferation. These results thus indicate that 3E8, C8
and G3 are the most useful among the 5 mAbs in reducing tumor cell
proliferation.
TABLE-US-00003 TABLE 3 Summary of signaling-blocking and
anti-proliferative activities of PRLR mAbs Abbre- Block Block Block
Block T-47D viated MAPK AKT STAT5 cell Clone name name Signaling
Signaling Signaling proliferation 4P1E9.C7 C7 - - - - 4P1B3.3E8 3E8
+++ +++ +++ +++ 4P1E5.2G5 2G5 ++ ++ ++ - 4P4F1.C8 C8 +++ +++ +++
+++ 4P4H6.G3 G3 +++ +++ +++ +++
Example 6
Pharmacokinetic (PK) Analysis of the Antibodies
[0260] PK analysis was performed as described in Materials and
Methods. The resulting PK statistics are shown in Table 4. AUCinf
indicates area under the curve to infinity, CL/F indicates
clearance, t1/2 indicates half-life in serum, Cmax indicates
maximal concentration, T max indicates time to reach maximal
concentration and Vdz/F indicates the volume of distribution.
TABLE-US-00004 TABLE 4 Pharmacokinetic (PK) Parameters of
antibodies in female athymic nude mice PK Parameters Unit 4P1E9.C7
4P1B3.3E8 4P1E5.2G5 4P4F1.C8 4P4H6.G3 AUCinf day*ug/mL 128.82
118.57 168.91 133.90 141.36 CL/F mL/day/kg 7.76 8.43 5.92 7.47 7.07
t1/2 day 8.51 11.18 9.98 8.19 10.26 Cmax mL/day/kg 9.21 11.10 11.46
10.62 9.92 Tmax day 4.00 0.25 1.00 1.00 0.25 Vdz/F mL/kg 95.33
136.00 85.21 88.24 104.76
[0261] Accordingly, the antibodies all had a quite long half-life
and, except C7, reached maximal concentrations rather quickly.
[0262] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention.
[0263] All of the publications, patents and patent applications
cited in this application are herein incorporated by reference in
their entirety to the same extent as if the disclosure of each
individual publication, patent application or patent was
specifically and individually indicated to be incorporated by
reference in its entirety.
Sequence CWU 1
1
11448PRTArtificial SequenceSynthetic construct 1Gln 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 Thr Thr Val Trp Glu Asn Leu Tyr Phe Gln Gly Val Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445
* * * * *