U.S. patent application number 13/584711 was filed with the patent office on 2013-01-10 for anti-hedgehog antibodies.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Suzanna J. Scales.
Application Number | 20130011853 13/584711 |
Document ID | / |
Family ID | 40433914 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130011853 |
Kind Code |
A1 |
Scales; Suzanna J. |
January 10, 2013 |
ANTI-HEDGEHOG ANTIBODIES
Abstract
The invention relates to anti-hedgehog antibodies, their use in
the detection of hedgehog expression in tissue, and to the use of
such detection in the treatment of cancer.
Inventors: |
Scales; Suzanna J.; (San
Mateo, CA) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
40433914 |
Appl. No.: |
13/584711 |
Filed: |
August 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13217191 |
Aug 24, 2011 |
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13584711 |
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12341910 |
Dec 22, 2008 |
8030454 |
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13217191 |
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61017232 |
Dec 28, 2007 |
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61099864 |
Sep 24, 2008 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C07K 16/18 20130101;
G01N 33/57496 20130101; A61P 37/04 20180101; G01N 33/57419
20130101; A61P 35/00 20180101; G01N 33/57449 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1-5. (canceled)
6. A method detecting hedgehog expression in tissue comprising the
steps of: (a) removing a tissue sample from a tumor in a patient
afflicted with the tumor; (b) contacting the tissue with an
anti-hedgehog antibody comprising a heavy chain and a light chain,
wherein the heavy chain comprises the antigen binding regions
HCHVR1 comprising the amino acid sequence of SEQ ID NO:2, HCHVR2
comprising the amino acid sequence of SEQ ID NO:4 and HCHVR3
comprising the amino acid sequence of SEQ ID NO:6 and the light
chain comprising the antigen binding regions LCHVR1 comprising the
amino acid sequence of SEQ ID NO:10, LCHVR2 comprising the amino
acid sequence of SEQ ID NO:12 and LCHVR3 comprising the amino acid
sequence of SEQ ID NO:14 wherein said anti-hedgehog antibody binds
to hedgehog polypeptide at an epitope within the region of amino
acid residues 70-96 and determining if hedgehog is overexpressed in
said tissue, compared to normal tissue; (c) measuring the extent of
binding.
7. The method of claim 6, wherein the tissue sample is
formalin-fixed paraffin-embedded FFPE prior to contacting with the
antihedgehog antibody.
8. The method of claim 7, wherein the detection method is selected
from the group consisting of IHC and Western blot.
9. A method for identifying tumors responsive to hedgehog
antagonists comprising the steps of: (a) removing a tissue sample
from a tumor in a patient afflicted with the tumor; (b) contacting
the tumor tissue or tissue proximal to the tumor with an
anti-hedgehog antibody comprising a heavy chain and a light chain,
wherein the heavy chain comprises the antigen binding regions
HCHVR1 comprising the amino acid sequence of SEQ ID NO:2, HCHVR2
comprising the amino acid sequence of SEQ ID NO:4 and HCHVR3
comprising the amino acid sequence of SEQ ID NO:6 and the light
chain comprising the antigen binding regions LCHVR1 comprising the
amino acid sequence of SEQ ID NO:10, LCHVR2 comprising the amino
acid sequence of SEQ ID NO:12 and LCHVR3 comprising the amino acid
sequence of SEQ ID NO:14 wherein said anti-hedgehog antibody binds
to hedgehog polypeptide at an epitope within the region of amino
acid residues 70-96 and determining if hedgehog is overexpressed in
said tissue, compared to normal tissue (c) contacting a normal
tissue sample with said anti-hedgehog antibody; (d) comparing the
levels of hedgehog in said tumor and said normal tissue
samples.
10. The method of claim 9, wherein the tissue sample is
formalin-fixed paraffin-embedded (FFPE) prior to contacting said
tissue sample with the anti-hedgehog antibody.
11. The method of claim 9, wherein the determination method is
selected from the group consisting of IHC and Western blot.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 13/217,191 filed Aug. 24, 2011 which is a divisional
application of U.S. Pat. No. 8,030,454, filed Dec. 22, 2008, which
claimed the benefit of priority to U.S. provisional application No.
61/017,232 filed Dec. 28, 2007 and U.S. provisional application No.
61/099,864 filed Sep. 24, 2008, the disclosures of which are all
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods of
diagnosing cancer and detecting protein expression in tumors. More
specifically, the invention relates to antibodies that bind to
mammalian hedgehog, and to their use in diagnosis and treatment of
conditions characterized by hedgehog expression, including
cancer.
BACKGROUND OF THE INVENTION
[0003] Members of the hedgehog family of signaling molecules
mediate many important short- and longrange patterning processes
during invertebrate and vertebrate embryonic, fetal, and adult
development. In Drosophila melanogaster, a single hedgehog gene
regulates segmental and imaginal disc patterning. In contrast, in
vertebrates, a hedgehog gene family (e.g., in mammals, Shh, Dhh,
Ihh, collectively "Hh") is involved in the control of
proliferation, differentiation, migration, and survival of cells
and tissues derived from all three germ layers, including, e.g.,
left-right asymmetry, CNS development, somites and limb patterning,
chondrogenesis, skeletogenesis and spermogenesis.
[0004] Hedgehog signaling occurs through the interaction of
hedgehog protein with the hedgehog receptor, Patched (Ptch), and
the co-receptor Smoothened (Smo). There are two mammalian homologs
of Ptch, Ptch-1 and Ptch-2 ("collectively "Ptch"), both of which
are 12 transmembrane proteins containing a sterol sensing domain
(Motoyama et al., Nature Genetics 18: 104-106 (1998), Carpenter et
al., P.N.A.S. (U.S.A.) 95 (23): 13630-40 (1998). The interaction of
Hh with Ptch triggers a signaling cascade that results in the
regulation of transcription by zinc-finger transcriptions factors
of the Gli family.
[0005] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease (Boring et al., CA
Cancel J. Clin. 43:7 (1993)). Cancer features one of more of the
following characteristics: (1) an the increase in the number of
abnormal, or neoplastic, cells derived from a normal tissue which
proliferate to form a tumor mass, (2) the invasion of adjacent
tissues by these neoplastic tumor cells, and (3) the generation of
malignant cells which eventually spread via the blood or lymphatic
system to regional lymph nodes and to distant sites via a process
called metastasis. In a cancerous state, a cell proliferates under
conditions in which normal cells would not grow. Cancer manifests
itself in a wide variety of forms, characterized by different
degrees of invasiveness and aggressiveness.
[0006] As often is the case when pathways that are active during
embryogenic development and mostly inactive in adults, reactivation
of hedgehog signaling has been implicated in a wide variety of
cancers and carcinogenesis. The earliest examples of Hh signaling
in cancers came from the discovery that Gorlin's syndrome, in which
patients frequently suffer basal cell carcinomas and are also
predisposed to medulloblastomas and rhabdomysocarcomas, is due to
an inactivating mutation in Ptch, resulting in Hh pathway
activation (Hahn et al 1998 Cell 85 p841; Johnson et al 1996,
Science 272 p1668). Subsequently inactivating mutations in Ptch HO
%) and or activating mutations in Smo (.about.10%) were found to be
responsible for sporadic basal cell carcinomas (Xie et al 1998,
Nature 391 p90).
[0007] Recently, it has become clear that another class of
Hh-associated cancers exist, which depend on Hh ligand secretion
from the tumor rather than mutational activation for pathway
activation. Such cancers include prostate, pancreatic and small
cell lung cancers (Watkins et al 2003, Nature 422 p313; Thayer et
al 2003 Nature 425 p851; Berman et al 2003 Nature 425 p846). A
subset of such cancers can be treated by Hh antagonists such as
small molecule antagonists of Smo or anti-Hh antibody 5E1 (Chen et
al 2002, PNAS 99 p14071; Williams et al 2003 PNAS 100 p4616; Rubin
and de Sauvage 2006 Nature Reviews Drug Discovery 5 p1026). While
not all Hh-expressing tumors respond to such antagonists, it is
very likely that those that do not express Hh will not respond;
indeed the Hh-negative DLD-1 colorectal xenograft model is not
inhibited by such treatment under conditions where Hh-positive
LS180, HT29 and HT55 tumor models are (Yauch/de Sauvage et al.
January 2008). As a result, there is a need for an effective
technique for determining hedgehog expression prior to application
of the hedgehog antagonists so as to identify hedgehog-secreting
tumors, in order to maximize the overall response rate.
[0008] Currently available antibodies that bind to mammalian
hedgehog (e.g., H160, Santa Cruz Biotech) are ineffective reagents
to detect the presence of hedgehog signaling because they do not
show sufficient sensitivity in the absence of background staining
This is particularly true on FFPE (formalin-fixed
paraffin-embedded) tissue specimens.
[0009] As a result, there is a need for antibodies that bind to
mammalian hedgehog (e.g., sonic hedgehog, Indian hedgehog and
desert hedgehog), particularly in FFPE specimens, for use to
detecting the expression of hedgehog both in diagnostic assays and
treatment regimens.
SUMMARY OF THE INVENTION
[0010] The invention provides for anti-hedgehog antibodies, and
their use in the detection of hedgehog expression and the treatment
of including hedgehog responsive cancer.
[0011] In one embodiment, the invention relates to an anti-hedgehog
antibody comprising a heavy chain and a light chain, wherein the
heavy chain comprises:
HCFR1-HCHVR1-HCFR2-HCHVR2-HCFR3-HCHVR3-HCFR4-CR and the light chain
comprises: LCFR1-LCCDR1-LCFR2-LCCDR2-LCFR3-LCCDR3-LCFR4. In a
specific aspect, the anti-Hh antibody comprises the heavy and light
chain sequences of 95.9.
[0012] In another embodiment, the invention relates to a method of
detecting hedgehog expression in tissue comprising contacting said
tissue with an anti-hedgehog antibody and measuring the extent of
binding, wherein said anti-hedgehog antibody binds to hedgehog
polypeptide at an epitope within the region of amino acid residues
75-96. In a specific aspect, the tissue is a tumor or cancer. In
yet another specific aspect, the tissue is removed from the host
prior to the determination. In a further specific aspect, the
tissue sample is FFPE prior the contacting with the anti-Hh
antibody. In a further specific aspect, the determination method is
selected from the group consisting of: of IHC and Western blot. In
a specific aspect, the binding sensitivity is greater than that of
the anti-Hh antibody H160. In a further specific aspect, the
anti-Hh antibody does not compete with ptch for binding to Shh. In
a further specific aspect, the anti-Hh is selected from the group
consisting of 95.3, 95.7 and 95.9.
[0013] In yet another embodiment, the invention relates to a method
for identifying tumors responsive to hedgehog antagonists,
comprising contacting the tumor tissue or tissue proximal to the
tumor with an anti-hedgehog antibody wherein said anti-hedgehog
antibody binds to hedgehog polypeptide at an epitope within the
region of amino acid residues 75-96 and determining if hedgehog is
overexpressed in said tissue, compared to normal tissue. In a
specific aspect, the tissue is removed from the host prior to the
determination. In yet another specific aspect, the tissue sample is
FFPE prior the contacting with the anti-Hh antibody. In a further
specific aspect, the determination method is selected from the
group consisting of: of IHC and Western blot. In a specific aspect,
the binding sensitivity is greater than that of the anti-Hh
antibody H160. In a further specific aspect, the anti-Hh antibody
does not compete with ptch for binding to Shh. In a further
specific aspect, the anti-Hh is selected from the group consisting
of 95.3, 95.7 and 95.9.
[0014] In a further embodiment, the invention relates to a cancer
treatment regimen in a patient comprising:
[0015] (a) contacting a tumor and/or tissue proximal to the tumor
removed from the patient with an anti-hedgehog antibody wherein
said anti-hedgehog antibody binds to hedgehog polypeptide at an
epitope within the region of amino acid residues 75-96,
[0016] (b) detecting the presence of hedgehog expression,
[0017] (c) comparing the hedgehog expression with that of normal or
tissue of the same type or origin not associated with the tumor,
and if such hedgehog is overexpressed,
[0018] (d) treating the patient with a hedgehog antagonist.
In a specific aspect, the tissue is removed from the host prior to
the determination. In another specific aspect, the tissue sample is
FFPE prior the contacting with the anti-Hh antibody. In yet another
specific aspect, the determination method is selected from the
group consisting of: of IHC and Western blot. In a further specific
aspect, the binding sensitivity is greater than that of the anti-Hh
antibody H160. In a further specific aspect, the anti-Hh antibody
does not compete with ptch for binding to Shh. In a further
specific aspect, the anti-Hh antibody is selected from the group
consisting of 95.3, 95.7 and 95.9.
[0019] In a further embodiment, the invention relates to an article
of manufacture (kit) for measuring hedgehog expression comprising
an anti-hedgehog antibody wherein said anti-hedgehog antibody binds
to hedgehog polypeptide at an epitope within the region of amino
acid residues 75-96, and instructions for determining if hedgehog
is overexpressed in a tissue, comprising contacting such tissue
with such anti-hedgehog antibody and measuring the extent of
binding. In a specific aspect, the tissue is a tumor or cancer. In
yet another specific aspect, the tissue is removed from the host
prior to the determination. In a further specific aspect, the
tissue sample is FFPE prior the contacting with the anti-Hh
antibody. In a further specific aspect, the determination method is
selected from the group consisting of: of IHC and Western blot. In
a specific aspect, the binding sensitivity is greater than that of
the anti-Hh antibody H160. In a further specific aspect, the
anti-Hh antibody does not compete with ptch for binding to Shh. In
a further specific aspect, the anti-Hh antibody is selected from
the group consisting of 95.3, 95.7 and 95.9.
[0020] In a further embodiment, the invention relates to a method
of screening patients with abnormal tissue growth for the risk of
developing cancer, comprising determining if such tissue
overexpresses hedgehog signaling with a hedgehog antibody wherein
said anti-hedgehog antibody binds to hedgehog polypeptide at an
epitope within the region of amino acid residues 75-96, comprising
contacting such tissue with such anti-hedgehog antibody and
measuring the extent of binding. In a specific aspect, the tissue
is a tumor or cancer. In yet another specific aspect, the tissue is
removed from the host prior to the determination. In a further
specific aspect, the tissue sample is FFPE prior the contacting
with the anti-Hh antibody. In a specific aspect, the binding
sensitivity is greater than that of the anti-Hh antibody H160. In a
further specific aspect, the anti-Hh antibody does not compete with
ptch for binding to Shh. In a further specific aspect, the anti-Hh
antibody is selected from the group consisting of 95.3, 95.7 and
95.9. In a further specific aspect, the determination method is
selected from the group consisting of IHC and Western Blot.
[0021] In a further embodiment, the invention relates to a method
of treating cancer comprising (a) contacting tissue suspected of
being cancerous or tissue proximal to such tissue with an
anti-hedgehog antibody wherein said anti-hedgehog antibody binds to
hedgehog polypeptide at an epitope within the region of amino acid
residues 750-96; (b) determining of hedgehog is overexpressed; and
(c) treating with a hedgehog antagonist. In yet another specific
aspect, the tissue is removed from the host prior to the
determination. In a further specific aspect, the tissue sample is
FFPE prior to contacting with the anti-Hh antibody. In a specific
aspect, the determination method is selected from the group
consisting of IHC and Western blot. In a specific aspect, the
binding sensitivity of the anti-hedgehog antibody is greater than
that of the anti-Hh antibody H160. In a further specific aspect,
the anti-Hh antibody does not compete with ptch for binding to Shh.
In a further specific aspect, the anti-Hh antibody is selected from
the group consisting of 95.3, 95.7 and 95.9.
DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A-F are micrographs showing the binding of parent
polyclonal anti-Shh antibody ("59 pAb") and its derived monoclonal
rMab specifically recognize Shh-transfected COS cells by
immunofluorescence (IF). Cos-7 cells with or without stably
transfected human Shh were fixed, permeabilized and stained with
rabbit antibodies, detected with Cy3-conjugated anti-human and
examined by fluorescence microscopy. 59A+B polyclonal on Shh-COS
(A) or untransfected (B) cells; C) H-160 on Shh-COS cells; D)
irrelevant rmAb on Shh-COS cells; purified 95.9 rMab on Shh-COS (E)
and untransfected (F) cells. Scale bar is 30 .mu.m.
[0023] FIGS. 2A-F are micrographs demonstrating that polyclonal
anti-Shh antibody ("59 pAb") and its derived rMAb specifically
recognize Shh-transfected 293 cells by IHC. FFPE sections of
untransfected (B, F) or human Shh transiently transfected (A, C, E)
293 cell pellets were processed for TARGET retrieval and stained
with the indicated rabbit antibodies, followed by HRP-anti rabbit
and detected with ABC-peroxidase Elite. A) irrelevant rabbit
antibody (R&D Systems) on Shh-293 cells; B) H-160 (Santa Cruz)
on Shh-293 cells; on Shh-293 cells; 59A+B affinity purified
polyclonal on Shh-293 (C) or untransfected (D) purified 95.9 rMab
on Shh 293 (E) and untransfected (F) cells. Samples A, B and D were
subjected to TSA-HRP amplification, resulting in some background
staining, so this step was 5 omitted from the remaining samples.
Scale bar is 50 .mu.m.
[0024] FIGS. 3A-L are micrographs showing that all of the
monoclonal antibodies 95.9, 95.7 and 95.3 also cross react with Ihh
and Dhh. (A) COS cells transfected with full length human Shh (top
row), Ihh (middle row) and Dhh (bottom row) were processed for
immunofluorescence as in FIG. 1, using affinity purified 95.3 (1-3)
95.7 (4-6), 95.9 (7-9) or H-160 (10-12) antibodies. Scale bar=30
.mu.m. (B) Western blot of 293 cells either untransfected (WT) or
transfected with human Dhh, Ihh or Shh full length proteins as
indicated and immunolabeled with 5 mg/ml purified 95.9 rMab (upper
panel) or H-160 (lower panel). Both antibodies recognize all three
full length (FL) Hh proteins (.apprxeq.50 kDa) as well as the
secreted Hh-N termini (.apprxeq.22 kDa), although 95.9 did not
recognize the .apprxeq.98 kDa background band that H-160 labeled in
untransfected cells. Molecular weight markers in kDa are shown on
the left.
[0025] FIGS. 4A-B show the N-terminal sequences of 95.9, 95.7 and
95.3. This strongly suggests that they are likely identical
subclones. A) N-terminal sequences of the light chains of the three
supposedly identical subclones of 95 are indeed identical in
positions where the sequence was unambiguous. The amino acid
residues in parentheses indicate ambiguous amino acids, dashes
indicate positions for which an amino acid was not determinable,
italic letters indicate discrepancies between the obtained
N-terminal sequence and the actual cloned sequence of 95.9
determined by DNA sequencing and conceptual translation (2.sup.nd
row). Amino acid position numbers (numbered with the missing signal
sequence starting at 1) are indicated in the top row of the table.
B) The N-terminal sequences of the 95 subclone heavy chains were
complicated by the presence of endogenous myeloma cell heavy chain,
thus two (partially mixed) sequences are shown for each subclone.
The presence of glutamine at position 20 is inferred.
[0026] FIG. 5 shows that 95.9 epitope maps to amino acids 76-90
within residues 24 to 197 of human sonic hedgehog (SEQ ID NO: 16).
95.9 coupled to agarose beads was incubated with recombinant Shh
and then subjected to trypsin digestion. The peptide was protected
from trypsin by 95.9 binding was eluted, identified by mass
spectrometry and confirmed by amino acid sequencing as amino acids
76-90, a region of Shh that is identical to residues 76-90 within
residues 24 to 197 of Ihh (SEQ ID NO: 17) and contains 3 amino acid
residues different to Dhh (SEQ ID NO: 18) (box region in the
alignment of the 3 Hh-N-terminal ligands, numbered with the first
amino acid residue of the signal sequence (not shown) designated as
1.
[0027] FIGS. 6A-F are micrographs showing, via IHC staining, that
95.9 recognizes endogenous Hh in the expected regions of developing
mouse embryos. A) 95.9 stains Hh in the ventral neural tube
floorplate (FP) and notochord (NC) of a transverse section of an
E10.5 mouse embryo much more strongly than the H-160 antibody under
the same conditions (both without TSA amplification) (B). C) E11.5
saggital section of neural tube floorplate (FP) and notochord (NC)
stained by 95.9. D) E11.5 embryo showing a possible diffusion
gradient of Hh from the ventral floor plate along the neural tube
(arrows). E) 95.9 stains the neuroepithelium of the developing
brain of an E11.5 mouse shown at lower magnification. TV,
telecephalic vesicle; 4th ventricle. F) E11.5 transverse section
through the developing mid-gut, showing the expected expression in
epithelial cells surrounding the lumen (arrow) and absence of
signal in the surrounding mesenchyme.
[0028] FIGS. 7A-E show that IHC staining by anti-Hh Ab 95.9
correlates well with transcript levels of Shh. 36 human colon
cancer cell lines were subjected to Q-PCR analysis of Shh mRNA and
IHC analysis (FFPE) using the purified 95.9 anti-Hh rMab in
parallel. Upper panel shows images of representative cell pellets
falling into each of the four IHC scoring categories as follows: A)
Hh-negative cell line, 1HC score 0+, although some non-specific
nuclear staining is discernible; B) IHC score 1+(weak staining), C)
cells illustrating 2+ IHC score (moderate staining), D) cells
showing strong staining (3+ IHC score). Scale bar is 50 .mu.m. FIG.
6E shows that relative Shh mRNA levels (Delta Ct values from Q-PCR
analysis) in each IHC score group, illustrating the trend towards
stronger 95.9 staining with lower Ct values (higher mRNA levels).
The Spearman coefficient of this correlation is -0.160, which is
statistically significant (p=0.0001).
[0029] FIGS. 8A-D show that 95.9 stains Shh+, but not Shh-, ovarian
cancer specimens. In situ hybridization of human ovarian cancer
specimens with an antisense probe to Shh shows one specimen
expresses Shh mRNA (black dots) in the tumor epithelium A) and the
other B) does not show any more signal than the sense probe
background (not shown). 95.9 staining of the same tumors shows
positive cytoplasmic and membranous signal (brown) in only the
Shh+tumor epithelium C), and not the Shh-negative specimen D).
Scale bar is 25 Om.
[0030] FIGS. 9A-B show that IHC staining of tumor TMA by anti-Hh Ab
95.9 detects different levels of Hh expression in colon, ovarian
and pancreatic tumors, respectively. Greater than 80% of CRC, OvCa
and PancCa express Hh ligands. A) Representative Hh-negative (0+),
low (1+), medium (2+) and high (3+) images are shown for colon (top
row), ovarian (middle row) and pancreatic (bottom row) tumors from
arrays of normal and tumor samples stained with the 95.9 antibody.
B) The total number of samples (n) stained is indicated for each
tumor type, and the number and percentages of the total examined
are indicated for 95.9 staining falling into each expression level
category.
[0031] FIGS. 10A-F show by IHC staining that anti-Hh Ab 95.9 is
sufficiently sensitive to detect low levels of Hh in hair
follicles. A) C57BL/6 4-week old mice skin labeled by 95.9 reveals
Shh signal in the hair follicles at 4 weeks of age when the hair is
in anagen phase. B) In situ hybridization of a longitudinal section
of fetal human skin (scalp) using an antisense Shh probe, showing
signal in the outer root sheath. Transverse section of fetal human
scalp stained with 95.9 (C) or H160 (D). Longitudinal sections of
fetal human scalp stained with 95.9 (E) or H160 (F), showing
staining in the proximal epithelium above the dermal papilla,
consistent with the ISH signal in (B). Scale-bar is 50 .mu.m in C,
D and 25 .mu.m in all other panels.
[0032] FIGS. 11A-B show the 95.9 amino acid sequences. The cloned
DNAs encoding 95.9's mature region heavy chain (A) or light chain
(B) (including Fc domain) was fused in frame with a generic
antibody signal sequence (box) and translated.
[0033] FIG. 12 shows Immunohistochemistry using mAb 95.5 and mAb
H-160 on mouse embryo E 10.5 neural tube, normal ovary (ISH-) and
ovarian cancer tissue (ISH+).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0034] "FFPE" means formalin fixed, paraffin embedded, which is
tissue resulting from a dissection or biopsy, that is then fixed in
order to prevent degeneration and to allow for histological,
pathological or cytological studies. This fixed tissue is then
embedded in the wax to cut its fine sections and then, to stain it
with Hemotoxylin and Eosin Stain, after which microtoming is
performed by cutting into fine sections. Fixation is the process by
which the tissue is immobilized, killed and preserved for further
study. Fixation makes tissue permeable to staining reagents and
cross-links its macromolecules so that they are stabilized and
locked in position. Any suitable fixatives may be used for this
purpose, and include for example, bouine solution, formaline, or
liquid nitrogen.
[0035] A "hedgehog responsive cancer" is a cancer or tumor that is
mediated by, or associated with hedgehog signaling such that the
presence of hedgehog (i.e., sonic hedgehog, indian hedgehog and/or
desert hedgehog) is necessary or essential for the survival and/or
progression of the cancer or tumor. Such hedgehog can be autocrine
(i.e., produced by the tumor itself) or paracrine, in which
hedgehog is produced by tissues in the proximity of the tumor or
cancer. In a specific aspect, a "hedgehog responsive cancer" is one
that is treated upon the application of a hedgehog antagonist.
[0036] The "overexpression" of hedgehog in a particular tissue or
tumor refers to hedgehog, such as polypeptide and/or nucleic acid
encoding such polypeptide, that is expressed at a level higher than
that which is present for non-diseased tissue of the same tissue
type or origin, or which is in the proximity of a tumor or cancer
that is a hedgehog responsive cancer, and is expressing hedgehog at
a higher level as compared with that which is expressed when a
hedgehog responsive cancer is not present, such as in a healthy, or
non-diseased state. Such overexpression may be caused by gene
amplification or by increased transcription or translation.
Hedgehog overexpression may be determined in a diagnostic or
prognostic assay by evaluating increased levels of the hedgehog
protein present on the surface of a cell, or secreted by the cell
(e.g., via an immunohistochemistry assay using the anti-hedgehog
antibodies of the invention. Alternatively, or additionally, one
may measure levels of hedgehog polypeptide-encoding nucleic acid or
mRNA in the cell, e.g., via fluorescent in situ hybridization using
a nucleic acid based probe corresponding to a hedgehog-encoding
nucleic acid or the complement thereof; (FISH; see WO98/45479
published October, 1998), Southern blotting, Northern blotting, or
polymerase chain reaction (PCR) techniques, such as real time
quantitative PCR (RT-PCR). One may also study hedgehog polypeptide
overexpression by measuring shed antigen in a biological fluid such
as serum, e.g., using antibody-based assays (see also, e.g., U.S.
Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr.
18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et
al., J. Immunol. Methods 132:73-80 (1990)). 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.
[0037] A "hedgehog antagonist" is an antibody, antigen binding
fragment thereof, other biological molecule or small molecule that
antagonizes or blocks hedgehog signaling, either by directly
binding to a hedgehog signaling pathway component and thereby
blocking the signal transduction through such component a
component, or by preventing the binding of a hedgehog signaling
pathway component to its natural binding partner so as to prevent
the transduction of a hedgehog signal.
[0038] The terms "hedgehog signaling pathway", "hedgehog pathway"
and "hedgehog signal transduction pathway" as used herein,
interchangeably refer to the signaling cascade mediated by hedgehog
and its receptors (e.g., patched, patched-2) and which results in
changes of gene expression and other phenotypic changes typical of
hedgehog activity. The hedgehog pathway may be activated in the
absence of hedgehog through activation of a downstream component
(e.g., overexpression of Smoothened or transfections with
Smoothened or Patched mutants to result in constitutive activation
with activate hedgehog signaling in the absence of hedgehog). The
transcription factors of the Gli family are often used as markers
or indicators of hedgehog pathway activation.
[0039] The term "Hh signaling component" refers to gene products
that participate in the Hh signaling pathway. An Hh signaling
component frequently materially or substantially affects the
transmission of the Hh signal in cells or tissues, thereby
affecting the downstream gene expression levels and/or other
phenotypic changes associated with hedgehog pathway activation.
[0040] Each Hh signaling component, depending on their biological
function and effects on the final outcome of the downstream gene
activation or expression, can be classified as either positive or
negative regulators. A positive regulator is an Hh signaling
component that positively affects the transmission of the Hh
signal, i.e., stimulates downstream biological events when Hh is
present. A negative regulator is an Hh signaling component that
negative affects the transmission of the Hh signal, i.e. inhibits
downstream biological events when Hh is present.
[0041] The binding of Hh to Ptch releases Smoothened (Smo), a 7
transmembrane G-coupled protein to then activate an intricate
intracellular signal-transduction pathway. The activation of Smo
then leads to signaling through a multimolecular complex, including
Costal2 (Cos2), Fused (Fu) and suppressor of Fused (Su(Fu)),
resulting in nuclear transport of the transcription factor Gli. Ho
et al., Curr. Opin. Neurobiol. 12:57-63 (2002); Nybakken et al.,
Curr. Opin. Genet. Dev. 12: 503-511 (2002); i Altaba et al., Nat.
Rev. Neurosci. 3: 24-33 (2002). There are three known Gli
transcription factors in verebrates: Gli1, Gli2 and Gli3. While
Gli1 is a transcriptional activator that is universally induced in
Hh-responsive cells, Gli2 and Gli3 can act either as activators or
repressors of transcription depending on the cellular context.
Absent Hh signaling, Gli3 is processed into a smaller, nuclear
transcriptional repressor that lacks the carboxy-terminal domain of
full-length Gli3. Upon activation of Smo, Gli3 protein cleavage is
prevented, and the full-length form with transcription-activation
function is generated. Gli2 also encodes a repressor function in
its carboxy-terminally truncated form, but its formation does not
appear to be regulated by Hh signaling. Stecca et al., J. Biol.
1(2):9 (2002).
Standard Definitions
[0042] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0043] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0044] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a hedgehog signaling or a
hedgehog signaling pathway component. Suitable hedgehog antagonist
antagonist molecules specifically include agonist or antagonist
antibodies or antibody fragments, fragments or amino acid sequence
variants of native hedgehog polypeptides, peptides, antisense
oligonucleotides, small organic molecules, etc. Methods for
identifying hedgehog antagonists may comprise contacting a cell in
which hedgehog signaling is active with a candidate antagonist
molecule and measuring a detectable change in one or more
biological activities normally associated with hedgehog signaling
(E.g., nuclear Gli expression).
[0045] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. A subject or mammal is
successfully "treated" for a hedgehog responsive cancer if, after
receiving a therapeutic amount of a hedgehog antagonist, the
patient shows observable and/or measurable reduction in, or absence
of one or more of the following: reduction in the number of cancer
cells or absence of the cancer cells; reduction in the tumor size;
inhibition (i.e., slow to some extent and preferably stop) of
cancer cell infiltration into peripheral organs including the
spread of cancer into soft tissue and bone; inhibition (i.e., slow
to some extent and preferably stop) of tumor metastasis;
inhibition, to some extent, of tumor growth; and/or relief to some
extent, one or more of the symptoms associated with the specific
cancer; reduced morbidity and mortality, and improvement in quality
of life issues. Reduction of these signs or symptoms may also be
felt by the patient.
[0046] The above parameters for assessing successful treatment and
improvement in the disease are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and other enzymes to determine spread to
the bone. CT scans can also be done to look for spread to the
pelvis and lymph nodes in the area. Chest X-rays and measurement of
liver enzyme levels by known methods are used to look for
metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0047] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0048] "Mammal" for purposes of the treatment of, alleviating the
symptoms of or diagnosis of a cancer refers to any animal
classified as a 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.
[0049] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0050] An "effective amount" of a hedgehog antagonist thereof as
disclosed herein is an amount sufficient to carry out a
specifically stated purpose. An "effective amount" may be
determined empirically and in a routine manner, in relation to the
stated purpose.
[0051] The term "therapeutically effective amount" refers to an
amount of a hedgehog antagonist effective to "treat" a disease or
disorder in a subject or mammal. In the case of cancer, especially
a hedgehog responsive cancer, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the cancer. See the definition
herein of "treating". To the extent the drug may prevent growth
and/or kill existing cancer cells, it may be cytostatic and/or
cytotoxic.
[0052] The term "package insert" 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.
Antibody Definitions
[0053] The term "antibody" includes monoclonal antibodies
(including full length antibodies which have an immunoglobulin Fc
region), antibody compositions with polyepitopic specificity,
multispecific antibodies (e.g., bispecific antibodies, diabodies,
and single-chain molecules, as well as antibody fragments (e.g.,
Fab, F(ab').sub.2, and Fv). The term "immunoglobulin" (Ig) is used
interchangeably with "antibody" herein.
[0054] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called a J chain, and contains 10 antigen binding sites, while IgA
antibodies comprise from 2-5 of the basic 4-chain units which can
polymerize to form polyvalent assemblages in combination with the J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain at its
other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L
is aligned with the first constant domain of the heavy chain
(C.sub.H1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see e.g., Basic and Clinical
Immunology, 8th Edition, Daniel P. Sties, Abba I. Ten and Tristram
G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page
71 and Chapter 6.
[0055] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (CH), immunoglobulins can be assigned to different classes
or isotypes. There are five classes of immunoglobulins: IgA, IgD,
IgE, IgG and IgM, having heavy chains designated .alpha., .delta.,
.epsilon., .gamma. and .mu., respectively. The .gamma. and .alpha.
classes are further divided into subclasses on the basis of
relatively minor differences in the CH sequence and function, e.g.,
humans express the following subclasses: IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2.
[0056] An "isolated" antibody is one that has been identified,
separated and/or recovered from a component of its production
environment (e.g., naturally or recombinantly). Preferably, the
isolated polypeptide is free of association with all other
components from its production environment. Contaminant components
of its production environment, such as that resulting from
recombinant transfected cells, are materials that would typically
interfere with research, diagnostic or therapeutic uses for the
antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. In preferred
embodiments, the polypeptide will be purified: (1) to greater than
95% by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments, to greater than 99% by weight; (1)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or
reducing conditions using Coomassie blue or, preferably, silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however, an
isolated polypeptide or antibody will be prepared by at least one
purification step.
[0057] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domains of the heavy chain and light
chain may be referred to as "VH" and "VL", respectively. These
domains are generally the most variable parts of the antibody
(relative to other antibodies of the same class) and contain the
antigen binding sites.
[0058] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines the
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
entire span of the variable domains. Instead, it is concentrated in
three segments called hypervariable regions (HVRs) both in the
light-chain and the heavy chain variable domains. The more highly
conserved portions of variable domains are called the framework
regions (FR). The variable domains of native heavy and light chains
each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three HVRs, which form loops
connecting, and in some cases forming part of, the beta-sheet
structure. The HVRs in each chain are held together in close
proximity by the FR regions and, with the HVRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies (see Kabat et al., Sequences of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in the binding of
antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibodydependent cellular
toxicity.
[0059] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations and/or post-translation modifications (e.g.,
isomerizations, amidations) that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site. In contrast to polyclonal antibody
preparations which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et
al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies
(see, e.g., Clackson et al., Nature, 352: 628 (1991); Marks et al.,
J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132
(2004), and technologies for producing human or human-like
antibodies in animals that have parts or all of the human
immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735;
WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:
2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);
Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0060] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding and/or the variable region
of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2 and Fv fragments; diabodies; linear antibodies
(see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein
Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and
multispecific antibodies formed from antibody fragments.
[0061] Papain digestion of antibodies produced two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having different antigenbinding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having a few additional
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0062] The Fc fragment comprises the carboxy-terminal portions of
both H chains 5 held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, the
region which is also recognized by Fc receptors (FcR) found on
certain types of cells.
[0063] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three HVRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0064] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the VH and VL antibody domains
connected into a single polypeptide chain. Preferably, the sFv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of the sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 20
(1994).
[0065] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody-variable domain that
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.
Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003)).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993) and Sheriff et al., Nature Struct. Biol. 3:733-736
(1996).
[0066] A number of HVR delineations are in use and are encompassed
herein. The HVRs that are Kabat complementarity-determining regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., supra). Chothia refers instead to the location
of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). The AbM HVRs represent a compromise between the Kabat CDRs
and Chothia structural loops, and are used by Oxford Molecular's
AbM antibody-modeling software. The "contact" HVRs are based on an
analysis of the available complex crystal structures. The residues
from each of these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0067] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and
26-35 (H1), 50-65 or 49-65 (a preferred embodiment) (H2), and
93-102, 94-102, or 95-102 (H3) in the VH. The variable-domain
residues are numbered according to Kabat et al., supra, for each of
these extended-HVR definitions.
[0068] "Framework" or "FR" residues are those variable-domain
residues other than the HVR residues as herein defined.
[0069] The expression "variable-domain residue-numbering as in
Kabat" or "amino-acid-position numbering as in Kabat," and
variations thereof, refers to the numbering system used for
heavy-chain variable domains or light-chain variable domains of the
compilation of antibodies in Kabat et agl., supra. Using this
numbering system, the actual linear amino acid sequence may contain
fewer or additional amino acids corresponding to a shortening of,
or insertion into, a FR or HVR of the variable domain. For example,
a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy-chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0070] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a VL or VH
framework derived from a human immunoglobulin framework or a human
consensus framework. An acceptor human framework "derived from" a
human immunoglobulin framework or a human consensus framework may
comprise the same amino acid sequence thereof, or it may contain
pre-existing amino acid sequence changes. In some embodiments, the
number of pre-existing amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less.
[0071] Alternatively, the framework and HVR definitions of the
anti-Hh antibody of the inventions may be defined as follows: (a)
the heavy chain may comprise:
HCFR1-HCHVR1-HCFR2-HCHVR2-HCFR3-HCHVR3-HCFR4-CR; wherein
HCFR1=QSVKESGGGLVQPEGSLTLTCTVS (SEQ ID NO: 1), HCHVR1=GFSLSSYDMS
(SEQ ID NO: 2 XM), HCFR2=WVRQAPGSGLEWI (SEQ ID NO: 3),
HCHVR2=GGILSGGSAYYASWAKS (SEQ ID NO: 4),
HCFR3=RSTITKNTNLNTVTLKMTSLTAADTATYFC (SEQ ID NO: 5),
HCHVR3=ARGIYPVGTNYNI (SEQ ID NO: 6), HCFR4=WGPGTLVTVSSG (SEQ ID NO:
7), CR QPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSV
RQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLG
GPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLR
EQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKV
YTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDN5
YKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSP GK (SEQ ID
NO: 8).
[0072] (b) the light chain may comprise:
LCFR1-LCCDR1-LCFR2-LCCDR2-LCFR3-LCCDR3-LCFR4; wherein
LCFR1=DIAVLTQTPSPVSAAVGGTVTINC (SEQ ID NO: 9), LCHVR1=QSSPSVYSNYLA
(SEQ ID NO: 10), LCFR2=WYQQKPGQPPKLLI (SEQ ID NO: 11),
LCHVR2=YYASTLAS (SEQ ID NO: 12),
LCFR3=GVPSRFKGSGSGTEFTLTISDLECADAATYYC (SEQ ID NO: 13),
LCHVR3=AGGYIDTSDTA (SEQ ID NO: 14),
LCFR4=FGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDG
TTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFN RGDC (SEQ
ID NO: 15).
[0073] An "amino-acid modification" at a specified position, e.g.
of the Fc region, refers to the substitution or deletion of the
specified residue, or the insertion of at least one amino acid
residue adjacent the specified residue. Insertion "adjacent" to a
specified residue means insertion within one to two residues
thereof. The insertion may be N-terminal or C-terminal to the
specified residue. The preferred amino acid modification herein is
a substitution.
[0074] An "affinity-matured" antibody is one with one or more
alterations in one or more HVRs thereof that result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody that does not possess those alteration(s). In
one embodiment, an affinity-matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity-matured
antibodies are produced by procedures known in the art. For
example, Marks et al., Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH- and VL-domain shuffling. Random
mutagenesis of HVR and/or framework residues is described by, for
example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813
(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.
Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896
(1992).
[0075] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope. For example, the
anti-Hh antibodies of the present invention specifically bind to Hh
(e.g., human sonic hedgehog (shh), human indian hedgehog (ihh) or
human desert hedgehog (dhh) and not to any other polypeptide.
[0076] A "blocking" antibody or an "antagonist" antibody is one
that inhibits or reduces a biological activity of the antigen it
binds. In some embodiments, blocking antibodies or antagonist
antibodies substantially or completely inhibit the biological
activity of the antigen. The anti-hedgehog antibodies of the
present invention are not blocking in the sense that once bound to
hedgehog, they do not prevent the binding of hedgehog so bound to
patched.
[0077] The term "solid phase" describes a non-aqueous matrix to
which the antibody of the present invention can adhere. Examples of
solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149. The term "Fc region" herein is used to define a
C-terminal region of an immunoglobulin heavy chain, including
native-sequence Fc regions and variant Fc regions. Suitable
native-sequence Fc regions for use in the antibodies of the
invention include human IgG1, IgG2, IgG3 and IgG4.
II. Hedgehog Antagonist Methods
[0078] Because hedgehog expression has been associated with
multiple physiological conditions and disease states, including
cancers that would be responsive to hedgehog antagonists, the
anti-hedgehog antibodies of the present invention are useful to
detect such events and disease states, as well as to identify such
responsive cancers.
[0079] A. Angiogenesis
[0080] Since hedgehog is known to stimulate angiogenesis, it is
expected that hedgehog antagonists, which inhibit hedgehog
activity, would be expected to inhibit angiogenesis, particularly
when some level of hedgehog signaling is a necessary perquisite for
angiogenesis. The anti-hedgehog antibodies of the invention can be
used to specifically identify tissues or conditions when hedgehog
expression is associated with angiogenesis. Angiogenesis is
fundamental to many disorders. Persistent, unregulated angiogenesis
occurs in a range of disease states, tumor metasteses and abnormal
growths by endothelial cells. The vasculature created as a result
of angiogenic processes supports the pathological damage seen in
these diseases.
[0081] Diseases associated with or resulting from angiogenesis
include: tumor growth, tumor metastasis or abnormal growths by
endothelial cells, including neovascular disease, age-related
macular degeneration, diabetic retinopathy, retinopathy of
prematurity, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasias, epidemic keratoconjuctivitis, Vitamin A
deficiency, contact lens overwear, atopic keratitis, superior
limbic keratitis, pterygium keratitis sicca, Sjogren's syndrome,
acne rosacea, phylctenulosis, syphilis, mycobacteria infections,
lipid degeneration, chemical burns, bacterial ulcers, fungal
ulcers, Herpes simplex infections, Herpes zoster infections,
protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's
marginal degeneration, marginal keratolysis, rheumatoid arthritis,
systemic lupus, polyarteritis, trauma, Wegener's granulomatosis,
sacroidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial
keratotomy, corneal graph rejection, rheumatoid arthritis, systemic
lupus, polyarteritis, trauma, Wegener's granulomatosis,
sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial
keratotomy, corneal graph rejection, rheumatoid arthritis,
osteoarthritis chronic inflammation (e.g., ulcerative colitis or
Crohn's disease), hemangioma, Osler-Weber Rendu disease, and
hereditary hemorrhagic telangiectasis.
[0082] Angiogenesis plays a critical role in cancer. A tumor cannot
expand without a blood supply to provide nutrients and remove
cellular wastes. Tumors in which angiogenesis is important include
solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing
sarcoma, neuroblastoma, osteosarcoma, and benign tumors such as
acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
Angiogenic factors have been found associated with several solid
tumors, and preventing angiogenesis could halt the growth of these
tumors and the resultant damage to the animal due to the presence
of the tumor. Angiogenesis is also associated with blood-born
tumors such as leukemias, any of various acute or chronic
neoplastic diseases of the bone marrow in which unrestrained
proliferation of white blood cells occurs, usually accompanied by
anemia, impaired blood clotting, and enlargement of the lymph
nodes, liver, and spleen. It is believed that angiogenesis plays a
role in the abnormalities in the bone marrow that give rise to
leukemia-like tumors.
[0083] In addition to tumor growth, angiogenesis is important in
metastasis. Initially, angiogenesis is important in the
vascularization of the tumor which allows cancerous cells to enter
the blood stream and to circulate throughout the body. After the
tumor cells have left the primary site, and have settled into the
secondary, metastatic site, angiogenesis must occur before the new
tumor can grow and expand. Therefore, prevention of angiogenesis
could lead to the prevention of metastasis of tumors and possibly
contain the neoplastic growth at the primary site.
[0084] Angiogenesis is also involved in normal physiological
processes such as reproduction and wound healing. Angiogenesis is
an important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis could be
used to induce amenorrhea, to block ovulation or to prevent
implantation by the blastula.
[0085] B. Disorders Resulting from Hyperactive Hedgehog
Signaling
[0086] The anti-hedgehog antibodies of the invention may be used to
determine is specific tissue and/or cells exhibit hedgehog
overexpression. This may occur in combination with other
measuresment of hedgehog pathway activation, such as through the
measurement of expression of gli genes activated by the hedgehog
signaling pathway. Gli-1, gli-2 and gli-3, most consistently
correlate with hedgehog signaling across a wide range or tissues
and disorders, while gli-3 is somewhat less so. The gli genes
encode transcription factors that activate expression of many genes
needed to elicit the full effects of hedgehog signaling. However,
the Gli-3 transcription factors can also act as a repressor of
hedgehog effector genes, and therefore, expression of gli-3 can
cause a decreased effect of the hedgehog signaling pathway. Whether
gli-3 acts as a transcriptional activator or repressor depends on
post-translational events, and therefore it is expected that
methods for detecting the activating form (versus the repressing
form) of Gli-3 protein would also be a reliable measure of hedgehog
pathway activation. The gli-1 gene is strongly expressed in a wide
array of cancers, hyperplasias and immature lungs, and serves as a
marker for the relative activation of the hedgehog pathway. In
addition, tissues such as immature lung, that have high gli gene
expression, are strongly affected by hedgehog inhibitors.
Accordingly, it is contemplated that the detection of gli gene
expression may be used as a powerful predictive tool to identity
tissues 5 and disorders that will particularly benefit from
treatment with a hedgehog antagonist.
[0087] Gli-1 expression levels are detected, either by direct
detection of the transcript or by detection of protein levels or
activity. Transcripts may be detected using any of a wide range of
techniques that depend primarily on hybridization or probes to the
gli-1 transcripts or to cDNAs synthesized therefrom. Well known
techniques include Northern blotting, reverse-transcriptase PCR and
microarray analysis of transcript levels. Methods for detecting Gli
protein levels include Western blotting, immunoprecipitation,
two-dimensional polyacrylamide gel electrophoresis (2D
SDS-PAGE--preferably compared against a standard wherein the
position of the Gli proteins has been determined), and mass
spectroscopy. Mass spectroscopy may be coupled with a series of
purification steps to allow high-throughput indentification of many
different protein levels in a particular sample. Mass spectroscopy
and 2D SDS-PAGE can also be used to identify post-transcriptional
modifications to proteins including proteolytic events,
ubiquitination, phosphorylation, lipid modification, etc. Gli
activity may also be assessed by analyzing binding to substrate DNA
or in vitro transcriptional activation of target promoters. Gel
shift assay, DNA footprinting assays and DNA-protein crosslinking
assays are all methods that may be used to assess the presence of a
protein capable of binding to Gli binding sites on DNA. J. Mol.
Med. 77(6):459-68 (1999); Cell 100(4): 423-34 (2000); Development
127(19): 4923-4301 (2000).
[0088] In certain embodiments, gli transcript levels are measured
and diseased or disordered tissues showing abnormally high gli
levels are treated with a hedgehog antagonist. In other
embodiments, the condition being treated is known to have a
significant correlation with aberrant activation of the hedgehog
pathway, even though a measurement of gli expression levels is not
made in the tissue being treated. Premature lung tissue, lung
cancers (e.g., adeno carcinomas, bronco-alveolar adenocarcinoma,
small cell carcinomas), breast cancers (e.g., inferior ductal
carcinomas, inferior lobular carcinomas, tubular carcinomas),
prostate cancers (e.g., adenocarcinomas), and benign prostatic
hyperplasias all show strongly elevated gli-1 expression levels in
certain cases. Accordingly, gli-1 expression levels are a powerful
diagnostic device to determine which of these tissues should be
treated with a hedgehog antagonist. In addition, there is
substantial correlative evidence that cancers of the urothelial
cells (e.g., bladder cancer, other urogenital cancers) will also
have elevated gli-1 levels in certain cases. For example, it is
known that loss of heterozygosity on chromosome 9q22 is common in
bladder cancers. The ptch-1 gene is located at this position and
ptch-1 loss of function is probably a partial cause of
hyperproliferation, as in many other cancer types. Accordingly,
such cancers would also show high gli expression and would be
particularly amenable to treatment with a hedgehog antagonist.
[0089] Expression of ptch-1 and ptch-2 is also activated by the
hedgehog signaling pathway, but not typically to the same extent as
gli genes, and as a result are inferior to the gli genes as markers
of hedgehog pathway activation. In certain tissues, only one of
ptch-1 or ptch-2 is expressed although the hedgehog pathway is
highly active. For example, in testicular development, desert
hedgehog plays an important role and the hedgehog pathway is
activated, but only ptc-2 is expressed. Accordingly, these genes
may be individually unreliable as markers for hedgehog pathway
activation, although simultaneous measurement of both genes is
contemplated as a more useful indicator for tissues to be treated
with a hedgehog antagonist.
[0090] Because gli is so ubiquitously expressed during hedgehog
activation, any degree of gli overexpression can also be useful in
combination with detection of hedgehog itself in determining that a
hedgehog antagonist will be an effective therapeutic. In such
embodiments, gli can be expressed at a level at least twice as high
as normal. In particular embodiments, expression is four, six,
eight or ten times as high as normal.
[0091] In light of the broad involvement of hedgehog signaling in
the formation of ordered spatial arrangements of differentiated
tissues in vertebrates, the hedgehog antagonists could be used in a
process for generating and/or maintaining an array of different
vertebrate tissue both in vitro and in vivo. The anti-hedgehog
antibodies can be used to identify when the application of a
hedgehog antagonist, whether inductive or anti-inductive with
respect to proliferation or differentiation of a given tissue type,
can be, as appropriate, any of the preparations described
above.
[0092] C. Neuronal Cell Culture
[0093] The hedgehog antibodies are further applicable to identify
cell culture techniques wherein reduction in hedgehog signaling,
and hence application of hedgehog antagonists is desirable. In
vitro neuronal culture systems have proved to be fundamental and
indispensable tools for the study of neural development, as well as
the identification of neurotrophic factors such as nerve growth
factor (NGF), ciliary trophic factors (CNTF), and brain derived
neurotrophic factor (BDNF). Once use of the present method may be
in culture of neuronal stem cells, such as in the use of such
cultures for the generation of new neurons and glia. These cultures
can be contacted with hedgehog antagonists in order to alter the
rate of proliferation or neuronal stem cells in the culture and/or
alter the rate of differentiation, or to maintain the integrity of
a culture of certain terminally differentiated neuronal cells. In
an exemplary embodiment, the subject method can be used to culture,
certain neuron types (e.g., sensory neurons, motor neurons). Such
neuronal cultures can be used as convenient assay systems as well
as sources of implantable cells for therapeutic treatments.
[0094] The anti-hedgehog antibodies of the present invention may be
applicable to a method of intracerebral grafting, an emerging
treatment for disorders of the central nervous system. For example,
one approach to repairing damaged brain tissues involves the
transplantation of cells from fetal or neonatal animals into the
adult brain. Dunnett et al., J. Exp. Biol. 123: 265-289 (1987).
Fetal neurons from a variety of brain regions can be successfully
incorporated into the adult brain, and such grafts can alleviate
behavioral defects. For example, movement disorder induced by
lesions of dopaminergic projections to the basal ganglia can be
prevented by grafts of embryonic dopaminergic neurons. Complex
cognitive functions that are impaired after lesions of the
neocortex can also be partially restored by grafts of embryonic
cortical cells. The subject method can be used to monitor the
regulation by hedgehog and/or hedgehog antagonists of the growth
state in a culture, or where fetal tissue is used, especially
neuronal stem cells, can be used to monitor the rate of
differentiation of the stem cells induced by hedgehog and/or
hedgehog antagonists.
[0095] Stem cells useful in the present invention are generally
known. For example, several neural crest cells have been
identified, some of which are multipotent and likely represent
uncommitted neural crest cells, and others of which can generate
only one type of cell, such as sensory neurons, and likely
represent committed progenitor cells. The anti-hedgehog antibodies
of the present invention can monitor the effectiveness of the
hedgehog antagonists applied to cultured stem cells, so as to
monitor the regulation of differentiation of the uncommitted
progenitor, or to monitor the regulation of the developmental fate
of a committed progenitor, or to monitor the regulation of the
developmental fate of a committed progenitor cell towards becoming
a terminally differentiated neuronal cell. For example, the present
method can be used in vitro to monitor the regulation of the
differentiation of neural crest cells into glial cells, schwann
cells, chromaffin cells, cholinergic, sympathetic or
parasympathetic neurons, as well as peptidergic and serotonergic
neurons. The hedgehog antagonist can be used alone, or in
combination with other neurotrophic factors that act to more
particularly enhance a particular differentiation fate of the
neuronal progenitor cell.
[0096] D. Regulation of Neuronal Growth and Differentiation
[0097] In addition to using the anti-hedgehog antibodies of the
present invention in combination with hedgehog antagonists in the
context of implantation of cell cultures, another aspect of the
present invention relates to monitoring the therapeutic application
of hedgehog antagonists to regulate the growth state of neurons and
other neuronal cells in both the central nervous system and the
peripheral nervous system. The ability of the hedgehog pathway
component (e.g., ptch, hedgehog, and smoothened) to regulate
neuronal differentiation during development of the nervous sytsem
and also presumably in the adult state indicates that in certain
instances, the subject hedgehog antagonists can be expected to
facilitate control of adult neurons with regard to maintenance,
functional performance, and aging of normal cells; repair and
regeneration processes in chemically or mechanically lesioned
cells; and treatment of degeneration in certain pathological
conditions. In light of this understanding, the present invention
specifically contemplated applications of the subject method to the
treatment (e.g., prevention, reduction in severity, etc.) of
neurological conditions deriving from: (i) acute, subacute, or
chronic injury to the nervous system, including traumatic injury,
chemical injury, vascular injury and deficits (such as the isehemia
resulting from stroke), together with infectious/inflammatory and
tumor-induced injury; (ii) aging of the nervous system, including
Parkinson's disease, Huntington's chorea, amyotrophic lateral
sclerosis and the like, as well as spinocerebellar degeneration;
and (iv) chronic immunological diseses of the nervous system or
affecting the nervous sytem, including multiple sclerosis.
[0098] As appropriate, the subject method can also be used in
generating nerve prosthesis for the repair of central and
peripheral nerve damage. In particular, where a crushed or severed
axon is intubulated by the use of a prosthetic device, hedgehog
antagonists can be added to the prosthetic device to regulate the
rate of growth and regeneration of the dendritic processes.
Exemplary nerve guidance channels are described in U.S. Pat. Nos.
5,092,871 and 4,955,892.
[0099] In another embodiment, the subject method can be used in the
treatment of neoplastic or hyperplastic transformation such as may
occur in the central nervous system. For instance, the hedgehog
antagonists can be utilitized to cause such transformed cells to
become either post-mitotic or apoptotic. The present method may,
therefore, be used as part of a treatment for, e.g., malignant
gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and
ependymomas.
[0100] E. Neuronal Cancer
[0101] The anti-hedgehog antibodies of the present invention can be
used in combination with hedgehog antagonists may be used as part
of a treatment regimen for malignant medulloblastoma and other
primary CNS malignant neuroectodermal tumors. Medulloblastoma, a
primary brain tumor, is the most common brain tumor in children. A
medulloblastoma is a primitive neuroectodermal (PNET) tumor arising
in the posterior fossa. They account for approximately 25% of all
pediatric brain tumors. Histologically, they are small round cell
tumors commonly arranged in true rosette, but may display some
differentiation to astrocytes, ependymal cells or neurons. PNETs
may arise in other areas of the brain including the penial gland
(pineoblastoma) and cerebrum. Those arising in the supratentorial
region generally have a worsened prognosis.
[0102] Medulloblastoma/PNETs are known to recur anywhere in the CNS
after resection, and can even metastasize to bone. Pretreatment
evaluation should therefore include and examination of the spinal
cord to exclude the possibility of "dropped metastases".
Gadolinium-enhanced MRI has largely replaced myelography for this
purpose, and CSF cytology is obtained postoperatively as a routine
procedure. The anti-hedgehog antibodies of the invention can used
to detect hedgehog expression levels in either primary, and/or
metastatic tumors, as part of treatment regimen to determine the
effectiveness of a hedgehog antagonist.
[0103] In other embodiment, the subject method is used as part of a
treatment program for ependymomas. Ependymomas account for
approximately 10% of the pediatric brain tumors in children.
Grossly, they are tumors that arise from the ependymal lining of
the ventricles and microscopically form rosettes, canals, and
perivascular rosettes. In the CHOP series of 51 children reported
with ependymomas, 3/4 were histologically benign. Approximately 2/3
arose from the region of the 4.sup.th ventricule. One third
presented in the supratentorial region. Age at presentation peaks
between birth and 4 years, as demonstrated by SEER data as well as
data from CHOP. The median age is about 5 years. Because so many
children with this disease are babies, they often require
multimodal therapy.
[0104] F. Non-Neuronal Cell Culture
[0105] The anti-hedgehog antibodies can be used in combination with
hedgehog antagonists in cell culture and therapeutic methods
relating to the generation and maintenance of non-neuronal tissue.
Such uses are contemplated as a result of the involvement of
hedgehog signaling components (e.g., ptch, hedgehog, smo, etc.) in
morphogenic signals of other vertebrate organogenic pathways, such
as endodermal patterning, and mesodermal and endodermal
differentiation.
[0106] Hedgehog signaling, especially ptc, hedgehog, and
smoothened, are involved in controlling the development of stem
cells responsible for formation of the digestive tract, liver,
lungs, and other organs derived from the primitive gut. Shh is the
inductive signal from the endoderm to the mesoderm, which is
critical to gut morphogenesis. Therefore, for example, the
anti-hedgehog antibodies of the instant method can be employed in
combination with hedgehog antagonists for regulating the
development and maintenance of an artificial liver that can have
multiple metabolic functions of a normal liver. In an exemplary
embodiment, the subject method can be used to monitor the
regulation by hedgehog antagonist of the functions of a normal
liver. For example, the subject method can be used to regulate the
proliferation and differentiation of digestive tube stem cells to
form hepatocyte cultures which can be used to populate
extracellular matrices, or which can be encapsulated in
biocompatible polymers, to form both implantable and extracorporeal
artificial livers.
[0107] In another embodiment, the subject method can be employed
therapeutically to monitor the regulation of such organs by
hedgehog antagonist after physical, chemical or pathological
insult. For instance, therapeutic comprising hedgehog antagonists
can be used in liver repair subsequent to a partial
hepactectomy.
[0108] In another embodiment, the subject method can be used to
monitor the proliferation and/or differentiation of pancreatic
tissue by hedgehog antagonists both in vivo and in vitro. The
generation of the pancreas and small intestine from the embryonic
gut depends on intercellular signaling between the endodermal and
mesodermal cells of the gut. In particular, the differentiation of
intestinal mesoderm into smooth muscle has been suggested to depend
on signals from adjacent endodermal cells. One candidate mediator
of endodermally derived signals in the embryonic hin dgut is Sonic
hedgehog (Shh). Apelqvist et al., Curr. Biol. 7: 801-4 (1997). The
Shh gene is expressed throughout the embryonic bud endoderm with
the exception of the pancreatic bud endoderm, which instead
expressed high levels of the homeodomain protein Ipf1/Pdx 1
(insulin promoter factor 1/pancreatic and duodenal homeobox 1), an
essential regulator of early pancreatic development. The Ipf1/Pdx1
was used to selectively express Shh in the developing pancreatic
epithelium. The pancreatic mesoderm of Ipf1/Pdx1-Shh transgenic
mice developed into smooth muscle and insterstitial cells of
Cajal--cells which are characteristic of the intestine, rather than
pancreatic mesenchyme and spleen. Apelqvist et al., supra. Also,
pancreatic explants exposed to Shh underwent as similar expression
of endodermally derived Shh controls the fate of adjacent mesoderm
at different regions of the gut tube.
[0109] In another embodiment, the anti-hedgehog antibodies hedgehog
antagonists are used to monitor the generation of endodermal tissue
from non-endodermal stem cells including mesenchymal cells and stem
cells derived from mesodermal tissues resulting from application of
hedgehog antagonist. Exemplary mesodermal tissues from which stem
cells may be isolated include skeletal muscle, cardiac muscle,
kidney, cartilage and fat.
[0110] G. Pancreatic Conditions/Disorders
[0111] There are a wide variety of pathological cell proliferative
and differentiative pancreatic conditions for which the
anti-hedgehog antibodies of the present invention when used in
combination with hedgehog antagonists may provide therapeutic
benefits. More specifically, such therapeutic benefits are directed
to correcting aberrant insulin expression, or modulation of
differentiation of pancreatic cells. More generally, however, the
present invention relates to a method of inducing and/or
maintaining a differentiated state, enhancing survival and/or
affecting proliferation of pancreatic cells, by contacting the
cells with the subject hedgehog antagonists. For instance, in light
of the apparent involvement of ptc, hedgehog and smoothened in the
formation of ordered spatial arrangements of pancreatic tissues,
the subject method could be used as part of a technique to monitor
the generation and/or maintenance of such tissue both in vitro and
in vivo. For instance, monitoring the modulation of hedgehog
signaling can be employed in both cell culture and therapeutic
methods involving generation and maintenance of .beta.-islet cells
and possibly also from nonpancreatic tissue, such as in controlling
the development and maintenance of tissue from the digestive tract,
spleen, lungs, urogenital organs (e.g., bladder), as well as other
organs which derive from the primitive gut.
[0112] In a specific embodiment, the anti-hedgehog antibodies of
the present invention, when used in combination with hedgehog
antagonists can be used in the treatment of hyperplastic and
neoplastic disorders affecting pancreatic tissue, especially those
characterized by aberrant proliferation of pancreatic cells. For
instance, pancreatic cancers are marked by abnormal proliferation
of pancreatic cells, which can result in alterations of insulin
secretory capacity of the pancreas. For instance, certain
pancreatic hyperplasias, such as pancreatic carcinomas, can result
in hypoinsulinemia due to dysfunction of .beta.-cells or decreased
islet cell mass. Moreover, manipulation of hedgehog signaling
properties at different points may be useful as part of a strategy
for reshaping/repairing pancreatic tissue both in vivo and in
vitro. In one embodiment, the present invention makes use of the
apparent involvement of ptc, hedgehog and smoothened in regulating
the development of pancreatic tissue. In another embodiment, the
subject antihedgehog antibodies, when used in combination with
hedgehog antagonists, can be employed therapeutically to regulate
the pancreas after physical, chemical or pathological insult. In
yet another embodiment, the subject method can be applied to cell
culture techniques, and in particular, may be employed to enhance
the initial integration of prosthetic pancreatic tissue devices.
Manipulation of proliferation and differentiation of pancreatic
tissue, such as through using hedgehog antagonists, can provide a
means for more carefully controlling the characteristics of a
cultured tissue. In an exemplary embodiment, the subject method can
be used to augment production of prosthetic devise which require
.beta.-islet cells, such as may be used in the encapsulation
devices described in, for example, as described in U.S. Pat. Nos.
4,892,538, 5,106,627, 4,391,909 and 4,353,888. Early progenitor
cells to the pancreatic islets are multipotential, and apparently
coactivate all the islet-specific genes from the time they first
appear. As development proceeds, expression of islet-specific
hormones, such as insulin, becomes restricted to the pattern of
expression characteristic of mature islet cells. The phenotype of
mature islet cells, however, is not stable in culture, as
reappearance of embryonal traits in mature .beta.-cells can be
observed. By utilizing the subject anti-hedgehog antibodies,
antagonists, one can monitor the differentiation path or
proliferative index of the cells regulated by hedgehog
antagonists.
[0113] Furthermore, monitoring the manipulation by hedgehog
antagonists of the differentiative state of pancreatic tissue can
be utilized in conjunction with transplantation of artificial
pancreas. For instance, manipulation of hedgehog function to affect
tissue differentiation can be utilized as a means of maintaining
graft viability.
[0114] H. Cell Proliferative Disorders, Tumors and Cancers
[0115] The anti-hedgehog antibodies the present invention may also
be used to combination with hedgehog antagonists to treat lung
carcinoma and adenocarcinoma, and other proliferative disorders
involving the lung epithelia. It is known that Shh is expressed in
human lung squamous carcinoma and adenocarcinoma cells. Fujita et
al., Biochem. Biophys. Res. Commun. 238: 658 (1997). The expression
of Shh was also detected in the human lung squamous carcinoma
tissues, but not in the normal lung tissue of the same patient. It
was also observed that Shh stimulates the incorporation of BrdU
into the carcinoma cells and stimulates their cell growth, while
anti-Shh-H inhibited such growth. These results suggest that a ptc,
hedgehog, and/or smoothened is involved in cell growth of such
transformed lung tissue and therefore indicates that detecting the
presence of hedgehog in such tissue may be determinative to
identify whether hedgehog antagonists can be used to treat such
lung carcinoma and adenocarcinomas, and other such proliferative
disorders involving the lung epithelia.
[0116] The anti-hedgehog antibodies hedgehog antagonists of the
present invention may also be used to identify tumors in which the
existence or pathogenesis is associated with hedgehog signaling,
and thus would be responsive to the application of hedgehog
antagonists. Such tumors include, but are not limited to: tumors
related to Gorlin's syndrome (e.g., medulloblastoma, meningioma,
etc.), tumors evidence in ptc knock-out mice (e.g., hemangioma,
rhabdomyosarcoma, etc.), tumors resulting from gli-1 amplification
(e.g., glioblastoma, sarcoma, etc.), tumors resulting from Smo
dysfunction (e.g., basal cell carcinoma, etc.), tumors connected
with TRC8, a ptc homolog (e.g., renal carcinoma, thyroid carcinoma,
etc.), Ext-1 related tumors (e.g., bone cancer, etc.), Shh-induced
tumors (e.g., lung cancer, chondrosarcomas, etc.), and other tumors
(e.g., breast cancer, urogenital cancer (e.g., kidney, bladder,
ureter, prostate, etc.), adrenal cancer, gastrointestinal cancer
(e.g., stomach, intestine, etc.).
[0117] The anti-hedgehog antibodies of the present invention may
also to identify cancer that would be responsive to the application
of hedgehog antagonists. These cancer include, but are not limited
to: prostate cancer, bladder cancer, biliary cancer, lung cancer
(including small cell and non-small cell), colon cancer, kidney
cancer, liver cancer, breast cancer, cervical cancer, endometrial
or other uterine cancer, ovarian cancer, testicular cancer, cancer
of the penis, cancer of the vagina, cancer of the urethra, gall
bladder cancer, esophageal cancer, or pancreatic cancer. Additional
cancer types include cancer of skeletal or smooth muscle, stomach
cancer, cancer of the small intestine, cancer of the salivary
gland, anal cancer, rectal cancer, thyroid cancer, parathyroid
cancer, pituitary cancer, and nasopharyngeal cancer. Further
exemplary forms of cancer which can be treated with the hedgehog
antagonists of the present invention include cancers comprising
hedgehog expressing cells. Still further exemplary forms of cancer
which can be treated with the hedgehog antagonists of the present
invention include cancers comprising gli expressing cells. In one
embodiment, the cancer is not characterized by a mutation in
patched-1.
[0118] K. Epithelial Tissue
[0119] The anti-hedgehog antibodies of the invention also may be
used to identify epithelial tissue that would be susceptible for
the therapeutic treatment (including prophylaxis) of disorders by
hedgehog antagonists. In general, such a treatment comprises
administering an amount of a hedgehog antagonist effective to alter
the growth state of the treated epithelial tissue that is
responsive to hedgehog signaling. The mode of administration and
dosage regimens will vary depending on the epithelial tissue(s)
that is to be treated (e.g., dermal, mucosal, glandular, etc.). In
a specific aspect, the method can be used to regulate the induction
of Shh induced differentiation and/or inhibit proliferation of
epithelially derived tissue. Thus, the anti-hedgehog antibodies of
the present invention can be used in a method for the treatment of
hyperplastic and/or neoplastic conditions involving epithelial
tissue that is responsive to hedgehog antagonists.
[0120] (1) Hair Growth
[0121] The anti-hedgehog antibodies of the invention can also be
used in combination with hedgehog antagonists control hair growth.
Hair is basically composed of keratin, a tough and insoluble
protein. Each individual hair comprises a cylindrical shaft and a
root, and is contained in a follicle, a flask-like depression in
the skin. The bottom of the follicle contains a finger-like
projection termed the papilla, which consists of connective tissue
from which hair grows, and through which blood vessels supply the
cells with nourishment. The shaft is the part that extends outwards
from the skin surface, whilst the root has been described as the
buried part of the hair. The base of the root expands into the hair
bulb, which rests upon the papilla. Cells from which the hair is
produced grow in the bulb of the follicle; they are extruded in the
form of fibers as the cells proliferate in the follicle. Hair
"growth" refers to the formation and elongation of the hair fiber
by the dividing cells.
[0122] As is well known in the art, the common hair cycle is
divided into three stages: anagen, catagen and telogen. During the
active phase (anagen), the epidermal stem cells of the dermal
papilla divide rapidly. Daughter cells move upward and
differentiate to form the concentric layers of the hair itself. The
transitional stage, catagen, is marked by the cessation of mitosis
of the stem cells in the follicle. The resting stage is known as
telogen, where the hair is retained within the scalp for several
weeks before an emerging new hair developing below it dislodges the
telogen-phase shaft from its follicle. From this model it has
become clear that the larger the pool of dividing stem cells that
differentiate into hair cells, the more hair growth occurs.
Accordingly, method for increasing or reducing hair growth can be
carried out by potentiating or inhibiting, respectively, the
proliferation of these stem cells.
[0123] The anti-hedgehog antibodies can be used to identify tissues
that would be responsive to the application of hedgehog antagonists
in a method of reducing the growth of human hair, either as a
replacement to or in combination with removal by cutting, shaving,
or depilation. For instance, the present method can be used in the
treatment of trichosis characterized by abnormally rapid or severe
growth of hair, e.g., hypertrichosis. In an exemplary embodiment,
hedgehog antagonists can be used to manage hirsutism, a disorder
marked by abnormal hairiness. The subject method can also provide a
process for extending the duration of depilation.
[0124] Moreover, because a hedgehog antagonist will often be
cytostatic to epithelial cells, rather than cytotoxic, such agents
can be used to protect hair follicle cells from cytotoxic agents
that require cell progression into S-phase of the cell-cycle for
efficacy, e.g., radiation-induced death. Treatment by the hedgehog
antagonists can provide protection by causing the hair follicle
cells to become quiescent, e.g., by preventing the cells from
entering S-phase, and thereby preventing the follicle cells from
undergoing mitotic catastrophe or programmed cells death. For
example, the hedgehog antagonists can be used in patients
undergoing chemo- or radiation-therapies that ordinarily result in
hair loss. By inhibiting cellcycle progression during such
therapies, the subject treatment can protect hair follicle cells
from death, which might otherwise result from activation of cell
death programs in the absence of quiescense. After therapy of the
hedgehog antagonists has concluded, the instant method can also be
removed with concomitant relief of the inhibition of follicle cell
proliferation. The anti-hedgehog antibodies of the invention can be
used to identify such tissue in which hedgehog signaling is active,
and hence would benefit from the application of hedgehog
antagonists.
[0125] The anti-hedgehog antibodies of the present invention can
also be used to identify patients suffering from folliculitis, such
as folliculitis decalvans, folliculitis ulerythematosis reticulate
or keloid folliculitis that would be responsive to hedgehog
antagonists. For example, a cosmetic preparation of a hedgehog
antagonist can be applied topically in the treatment of
pseudofolliculitis, a chronic disorder occurring most often in the
submandibular region of the neck and associated with shaving, the
characteristic lesions of which are erythematous papules and
pustules containing buried hairs.
[0126] The hedgehog antagonists of the invention can be used to
identify tissues that would be responsive to hedgehog antagonists
in a method of modulating the growth of human hair. Sato et al., J.
Clin. Invest. 104: 855-864 (1999) reported that upregulation of Shh
activity in postnatal skin functions as a biologic switch that
induces resting hair follicles to enter anagen with consequent hair
growth. Sato et al., used an adenovirus vector, AdShh, to transfer
the murine Shh cDNA to skin of postnatal day 19 C57BL/6 mice. The
treated skin showed increased mRNA expression of Shh, Patched, and
Gli-1. In mice receiving AdShh, but not in controls, acceleration
into anagen was evident, since hair follicle size and melanogenesis
increased and the hair-specific keratin ghHb-1 and the melanin
synthesis-related tyrosinase mRNAs accumulated. Finally, C57BL/6
mice showed marked acceleration of the onset of new hair growth in
the region of AdShh administration to skin weeks after treatment,
but not in control vector-treated or untreated areas. After 6
months, AdShh-treated skin showed normal hair and normal skin
morphology. Thus, the anti-hedgehog antibodies the present
invention may be useful to identify when application of hedgehog
antagonists would be useful to regulate or modulate Shh-induced
hair growth.
[0127] (2) Excessive Epithelial Proliferation
[0128] The anti-hedgehog antibodies of the present invention can be
used to identify hyperplastic conditions (e.g., keratosis) and
neoplastic epidermal conditions characterized by a high
proliferation rate (e.g., squamous cell carcinoma) that would be
responsive to hedgehog antagonists. This includes the treatment of
autoimmune diseases affecting the skin, in particular, or
dermatological diseases involving morbid proliferation and/or
keratinization of the epidermis, as for example, caused by
psoriasis or atopic dermatosis. These anti-hedgehog antibodies
could also be used to identify common skin disorders that are
characterized by localized abnormal proliferation of the skin
(e.g., psoriasis, squamous cell carcinoma, keratocanthoma, actinic
keratosis) which would be expected to be treatable by application
of the hedgehog antagonists.
[0129] The anti-hedgehog antibodies of the invention can also be
used to identify variety of other disorders characterized by
keratotic lesions that would be suitable for treatment with
hedgehog antagonists. Actinic keratoses, for example, are
superficial inflammatory premalignant tumors arising on sun-exposed
and irradiated skin. Accordingly, treatment of keratosis, such as
actinic keratosis, includes application of a hedgehog antagonist
composition in amounts sufficient to inhibit hyperproliferation of
epidermal/epidermoid cells of the lesion.
[0130] (3) Acne
[0131] Acne represents yet another dermatologic ailment which may
be treated by the hedgheg antagonists. Acne vulgaris, a multifactor
disease most commonly occurring in teenagers and young adults, is
characterized by the appearance of inflammatory and noninflammatory
lesions on the face and upper trunk. The basic defect which gives
rise to acne vulgaris is hypercornification of the duct of a
hyperactive sebaceous gland. Hypercornification blocks the normal
mobility of skin and follicle microorganisms, and in so doing,
stimulates the release of lipases by Propinobacterium acnes and
Staphylococcus epidermidis bacteria and Pitrosporum ovale, a yeast.
The anti-hedgehog antibodies of the present invention may be used
to identify patients or tissues in which treatment with hedgehog
antagonists would be effective. In particular, topical
preparations, may be useful for preventing the transitional
features of the ducts, e.g., hypercornification, which lead to
lesion formation. Such a therapeutic regimen comprising hedgehog
antagonists may further include in additional components, such as
for example, antibiotics, retinoids and antiandrogens.
[0132] (4) Dermatitis and Other Skin Ailments
[0133] The anti-hedgehog antibodies of the present invention may
also be used to identify patients or tissues suffering from
dermatitis that would be responsive to hedgehog antagonists.
Dermatitis is a descriptive term referring to poorly demarcated
lesions that are either pruritic, erythematous, scaly, blistered,
weeping, fissured or crusted. These lesions arise from any of a
wide variety of causes. The most common types of dermatitis are
atopic, contact and diaper dermatitis. For example, seborrheic
dermatitis is a chronic, usually pruritic, dermatitis with
erythema, dry, moist, or greasy scaling, and yellow-crusted patches
on various areas, especially the scalp, with exfoliation of an
excessive amount of dry scales. Hedgehog antagonists may also be
used in the treatment of stasis dermatitis, an often chronic,
usually eczematous dermatitis. Actinici dermatitis is a dermatitis
that due to exposure to actinic radiation such as that from the
sun, ultraviolet waves, or x- or gamma-radiation. According to the
present invention, the subject method can be used in the treatment
and/or prevention of certain symptoms of dermatitis caused by
unwanted proliferation of epithelial cells. Such therapies for
these various forms of dermatitis can also include topical and
systemic corticosteroids, antipruritics, and antibiotics.
[0134] Additional ailments that may be treated by the subject
method are disorders specific to non-humans, such as mange.
[0135] L. Non-Canonical Hedgehog Signaling
[0136] The anti-hedgehog antibodies of the present invention can be
used to identify situations or conditions in which hedgehog
antagonists can be used to regulate the activity in a noncanonical
Shh pathway that is independent of the Patched-Smoothened receptor
complex and the Gli transcription factors. In a recent report,
Jarov et al., Dev. Biol. 261(2): 520-536 (2003), describes that,
when Shh was immobilized to the substrate (extracellular matrix) or
produced by neuroepithelial cells themselves after transfection,
neural plate explants failed to disperse and instead formed compact
structures. Changes in the adhesive capacities of neuroepithelial
cells caused by Shh could be accounted for by inactivation of
surface .beta.1-integrins combined with an increase in
N-cadherin-mediated cell adhesion. This immobilized-Shh-mediated
adhesion does not contradict or interfere with the previously known
(soluble) Shh-mediated inductive, mitogenic, and trophic functions,
since the immobilized Shh promoted differentiation of
neuroepithelial cells into motor neurons and floor plate cells with
the same potency as soluble Shh. It has also been demonstrated that
Shh-regulation of adhesion properties during neural tube
morphogenesis is rapid and reversible, and it does not involve the
classical Patched-Smoothened-Gli signaling pathway, and it is
independent and discernible from Shh-mediated cell differentiation.
Thus, modifications of the adhesive properties of neural epithelial
cells induced by Shh cannot be attributed to its differentiation
promoting effect, but reveal a novel function of Shh in this tissue
that has not been described previously. Thus, the anti-hedgehog
antibodies of the present invention can identify conditions where
hedgehog antagonists of the present invention may be used to
regulate this non-canonical hedgehog pathway that is independent of
Ptch, Smo, Fu, Su(Fu) and/or Gli. More specifically, such hedgehog
antagonists may be used in a method to disrupt this function in
neuronal or other applicable tissues, preferably at specific
developmental stages.
III. Compositions and Methods
[0137] A. Anti-Hedgehog Antibodies
[0138] Exemplary antibodies that may be used for such purposes
include polyclonal and monoclonal antibodies. The term "antibodies"
sometimes also include antigen-binding fragments.
[0139] 1. Polyclonal Antibodies
[0140] 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, SOCl2, or R1N.dbd.C=NR, where R
and R1 are different alkyl groups.
[0141] Animals are 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.
[0142] 2. Monoclonal Antibodies
[0143] 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 (U.S. Pat. No. 4,816,567).
[0144] 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 or are capable of producing
antibodies that will specifically bind 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 (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0145] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0146] 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 available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0147] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, 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).
[0148] 3. Antibody Fragments
[0149] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies.
[0150] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now 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). See WO 93/16185; U.S. Pat.
No. 5,571,894; and U.S. Pat. No. 5,587,458. 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. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g.,
as described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0151] B. Variants of Anti-Hedgehog Antibodies
[0152] In addition to the anti-hedgehog antibodies described
herein, it is contemplated that variants of such molecules can be
prepared for use with the invention herein. Such variants can be
prepared by introducing appropriate nucleotide changes into the
encoding DNA, and/or by synthesis of the desired antibody or
polypeptide. Those skilled in the art will appreciate that amino
acid changes may alter posttranslational processes of these
molecules, such as changing the number or position of glycosylation
sites so as to enhance the hedgehog binding properties. Variations
in amino acid sequence can be made, for example, using any of the
techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S. Pat. No. 5,364,934.
Variations may be a substitution, deletion or insertion of one or
more codons encoding the amino acid sequence that results in a
change in the amino acid sequence as compared with the native
sequence. Optionally the variation is by substitution of at least
one amino acid with any other amino acid in one or more of the
domains of the amino acid sequence of interest. Guidance in
determining which amino acid residue may be inserted, substituted
or deleted without adversely affecting the desired activity may be
found by comparing the sequence of the amino acid sequence of
interest with homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high
homology. Amino acid substitutions can be the result of replacing
one amino acid with another amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine
with a serine, i.e., conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1
to 5 amino acids. The variation allowed may be determined by
systematically making insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for
activity exhibited by the full-length or mature native
sequence.
[0153] Fragments of the various anti-hedgehog antibodies are
provided herein. Such fragments may be truncated at the N-terminus
or C-terminus, or may lack internal residues, for example, when
compared with a full length native antibody or protein. Such
fragments which lack amino acid residues that are not essential for
a desired biological activity are also useful with the disclosed
methods.
[0154] The above polypeptide fragments may be prepared by any of a
number of conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
such fragments by enzymatic digestion, e.g., by treating the
protein with an enzyme known to cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with
suitable restriction enzymes and isolating the desired fragment.
Yet another suitable technique involves isolating and amplifying a
DNA fragment encoding the desired fragment fragment by polymerase
chain reaction (PCR). Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers
in the PCR. Preferably, such fragments share at least one
biological and/or immunological activity with the corresponding
full length molecule.
[0155] In particular embodiments, conservative substitutions of
interest are shown in 5 Table 1 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 1, or as further described below
in reference to amino acid classes, are introduced and the products
screened in order to identify the desired variant.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu; Asn Glu
Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly
(G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met;
Ala; Phe; Norleucine Leu Leu (L) Norleucine, Ile; Val; Met; Ala;
Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F)
Trp; Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr
Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;
Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0156] Substantial modifications in function or immunological
identity of the anti-hedgehog antibody variants are accomplished by
selecting substitutions that differ significantly in their effect
on maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral
hydrophilic: Cys, Ser, Thr; Asn; Gln (3) acidic: Asp, Glu; (4)
basic: H is, Lys, Arg; (5) residues that influence chain
orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
[0157] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0158] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the anti-hedgehog antibody molecule.
[0159] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244:1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0160] Any cysteine residue not involved in maintaining the proper
conformation of the anti-hedgehog antibody variant 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 such a molecule to
improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).
[0161] A particularly preferred type of substitutional variant
involves substituting one or more residues of a binding region of
the antibody. For example, in the case of an anti-hedgehog
antibody, a 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 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) as herein disclosed. 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 target
polypeptide. 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.
[0162] Nucleic acid molecules encoding amino acid sequence variants
of anti-hedgehog antibody variants are prepared by a variety of
methods known in the art. These methods include, but are not
limited to, isolation from a natural source (in the case of
naturally occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of a native sequence or an
earlier prepared variant.
[0163] C. Preparation of Anti-Hedgehog Antibodies
[0164] The description below relates primarily to production of
anti-hedgehog antibodies by culturing cells transformed or
transfected with a vector containing nucleic acid encoding such
antibodies. It is, of course, contemplated that alternative
methods, which are well known in the art, may be employed to
prepare such antibodies. For instance, the appropriate amino acid
sequence, or portions thereof, may be produced by direct peptide
synthesis using solid-phase techniques [see, e.g., Stewart et al.,
Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco,
Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)].
In vitro protein synthesis may be performed using manual techniques
or by automation. Automated synthesis may be accomplished, for
instance, using an Applied Biosystems Peptide Synthesizer (Foster
City, Calif.) using manufacturer's instructions. Various portions
of such antibodies may be chemically synthesized separately and
combined using chemical or enzymatic methods to produce the desired
product.
[0165] 1. Selection and Transformation of Host Cells
[0166] Host cells are transfected or transformed with expression or
cloning vectors described herein for anti-hedgehog antibody
production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. The culture
conditions, such as media, temperature, pH and the like, can be
selected by the skilled artisan without undue experimentation. In
general, principles, protocols, and practical techniques for
maximizing the productivity of cell cultures can be found in
Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.
(IRL Press, 1991) and Sambrook et al., supra.
[0167] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing 5 DNA into cells,
such as by nuclear microinjection, electroporation, bacterial
protoplast fusion with intact cells, or polycations, e.g.,
polybrene, polyornithine, may also be used. For various techniques
for transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0168] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include 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.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kanr 25; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0169] Full length antibody and antibody fragments can be produced
in bacteria, in particular when glycosylation and Fc effector
function are not needed. Full length antibodies have greater half
life in circulation. Production in E. coli can be faster and more
cost efficient. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237
(Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), and U.S.
Pat. No. 5,840,523 (Simmons et al.) which describes translation
initiation region (TIR) and signal sequences for optimizing
expression and secretion, these patents incorporated herein by
reference. 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. Final
purification can be carried out similar to the process for
purifying antibody expressed in suitable cells (e.g., CHO
cells).
[0170] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for vectors encoding hedgehog polypeptides. Saccharomyces
cerevisiae is a commonly used lower eukaryotic host microorganism.
Others include Schizosaccharomyces pombe (Beach and Nurse, Nature,
290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces
hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology,
9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]),
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; Van den Berg et al., Bio/Technology, 8:135 (1990)),
K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,
28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,
76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces
occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous
fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.
nidulans (Ballance et al., Biochem. Biophys. Res. Commun.,
112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton
et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A.
niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of specific species that are
exemplary of this class of yeasts may be found in C. Anthony, The
Biochemistry of Methylotrophs, 269 (1982).
[0171] Suitable host cells for the expression of glycosylated
hedgehog kinase polypeptide production are derived from
multicellular organisms. Examples of invertebrate cells include
insect cells such as Drosophila S2 and Spodoptera Sf9, as well as
plant cells, such as cell cultures of cotton, corn, potato, 25
soybean, petunia, tomato, and tobacco. Numerous baculoviral strains
and variants and corresponding permissive insect host cells from
hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (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.
[0172] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. 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, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); 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 (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 5 cells; and a human hepatoma line (Hep G2).
[0173] Host cells are transformed with the above-described
expression or cloning vectors for antihedgehog antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0174] 2. Selection and Use of a Replicable Vector
[0175] The nucleic acid (e.g., cDNA or genomic DNA) encoding the
respective anti-hedgehog antibody may be inserted into a replicable
vector for cloning (amplification of the DNA) or for expression.
Various vectors are publicly available. The vector may, for
example, be in the form of a plasmid, cosmid, viral particle, or
phage. The appropriate nucleic acid sequence may be inserted into
the vector by a variety of procedures. In general, DNA is inserted
into an appropriate restriction endonuclease site(s) using
techniques known in the art. Vector components generally include,
but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan.
[0176] The light and heavy chains of the anti-hedgehog antibody may
be expressed not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which may be a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. In general, the signal
sequence may be a component of the vector, or it may be a part of
the DNA encoding the mature sequence that is inserted into the
vector. The signal sequence may be a prokaryotic signal sequence
selected, for example, from the group of the alkaline phosphatase,
penicillinase, 1 pp, or heatstable enterotoxin II leaders. For
yeast secretion the signal sequence may be, e.g., the yeast
invertase leader, alpha factor leader (including Saccharomyces and
Kluyveromyces a-factor leaders, the latter described in U.S. Pat.
No. 5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders.
[0177] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0178] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0179] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up nucleic acid encoding the desire protein, such as DHFR
or thymidine kinase. An appropriate host cell when wild-type DHFR
is employed is the CHO cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub et al., Proc. Natl.
Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use
in yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0180] Expression and cloning vectors usually contain a promoter
operably linked to the nucleic acid sequence encoding the desired
amino acid sequence, in order to direct mRNA synthesis. Promoters
recognized by a variety of potential host cells are well known.
Promoters suitable for use with prokaryotic hosts include the
.beta.-lactamase and lactose promoter systems [Chang et al.,
Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],
alkaline phosphatase, a tryptophan (tip) promoter system [Goeddel,
Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters
such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.
USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also
will contain a Shine-Dalgarno (S.D.) Sequence operably linked to
the DNA encoding the desired protein sequence.
[0181] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0182] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0183] DNA Transcription in mammalian host cells is controlled, for
example, by promoters obtained from the genomes of viruses such as
polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, and from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0184] Transcription of a DNA encoding the anti-hedghog antibody
may be increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp, that act on a promoter to increase its transcription. Many
enhancer sequences are now known from mammalian genes (globin,
elastase, albumin, .alpha.-fetoprotein, and insulin). Typically,
however, one will use an enhancer from a eukaryotic cell virus.
Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or 3' to the coding sequence of the
preceding amino acid sequences, but is preferably located at a site
5' from the promoter.
[0185] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
respective antibody, polypeptide or oligopeptide described in this
section.
[0186] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of the respective antibody, polypeptide
or oligopeptide in recombinant vertebrate cell culture are
described in Gething et al., Nature, 293:620-625 (1981); Mantei et
al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
[0187] 3. Culturing the Host Cells
[0188] The host cells used to produce the anti-hedgehog antibodies
may be cultured in a variety of media. Commercially available media
such as Ham'S F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0189] 4. Detecting Gene Amplification/Expression
[0190] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0191] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies suitable for the
present method may be prepared against a native sequence
polypeptide or against exogenous sequence fused to DNA and encoding
a specific antibody epitope of such a polypeptide or
oligopeptide.
[0192] 5. Purification
[0193] Anti-Hedgehog antibodies may be recovered from culture
medium or from host cell lysates. If membrane-bound, it can be
released from the membrane using a suitable detergent solution
(e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in
expression of the preceding can be disrupted by various physical or
chemical means, such as freeze-thaw cycling, sonication, mechanical
disruption, or cell lysing agents.
[0194] It may be desirable to purify the preceding from recombinant
cell proteins or polypeptides. The following procedures are
exemplary of suitable purification procedures: by fractionation on
an ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex.RTM. G-75; protein A
Sepharose.RTM. columns to remove contaminants such as IgG; and
metal chelating columns to bind epitope-tagged forms of the desired
molecules. Various methods of protein purification may be employed
and such methods are known in the art and described for example in
Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular
antibody, polypeptide or oligopeptide produced for the claimed
methods.
[0195] When using recombinant techniques, the anti-hedgehog
antibodies can be produced intracellularly, in the periplasmic
space, or directly secreted into the medium. If such molecules are
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon.RTM. or
Millipore ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0196] Purification can occur 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.1, .gamma.2 or
.gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13
(1983)). Protein G is recommended for all mouse isotypes and for
human .gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)). 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.RTM. 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.
[0197] 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).
[0198] D. Articles of Manufacture and Kits
[0199] For therapeutic applications, the article of manufacture
comprises a container and a label or package insert on or
associated with the container indicating a use for the detection of
hedgehog expression, including in combination with a hedgehog
antagonist. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds the
composition comprising the anti-hedgehog antibody and may have a
sterile access port. The label or package insert indicates that the
composition is used for detecting hedgehog expression. The label or
package insert will further comprise instructions for using the
anti-hedgehog antibody.
[0200] Kits may also be provided that are useful for various other
purposes, e.g., for hedgehog-expressing cell killing assays, for
purification or immunoprecipitation of hedgehog from cells. For
isolation and purification of hedgehog kinase polypeptide, the kit
can contain the respective hedgehog kinase-binding reagent coupled
to beads (e.g., Sepharose.RTM. beads). Kits can be provided which
contain such molecules for detection and quantitation of hedgehog
polypeptide in vitro, e.g., in an IHC, ICC, ISH, EIA, ELISA or a
Western blot. As with the article of manufacture, the kit comprises
a container and a label or package insert on or associated with the
container. The container holds a composition comprising at least
one such anti-hedgehog antibody molecule useable with the
invention. Additional containers may be included that contain,
e.g., diluents and buffers, control antibodies. The label or
package insert may provide a description of the composition as well
as instructions for the intended in vitro or diagnostic use.
EXAMPLES
Example 1
Preparation of Anti-Hh Antibodies
Antigen Preparation:
[0201] The N-terminus of human Shh (aa 24-197) was subcloned into
the pST293 vector with an N terminal unizyme histidine (HQ) tag in
place of the native signal sequence under a phoA promoter and
transformed into 58F3 E. Coli. A starter culture was diluted 1:100
in 500 ml C.R.A.P. phosphate limiting media with 50 .mu./ml
carbencillin for 24 h at 30.degree. C. The phoA promoter induces
protein expression once phosphate is depleted from the media at an
OD.sub.600 of .about.2 (about 7 h later), at which time 100 .mu.M
zinc sulfate was added to help Shh folding. The cell pellet was
resuspended in 5 volumes (w/v) of lysis buffer (25 mM sodium
phosphate pH 8.0, 0.15 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 mM
B-mercaptoethanol) with a Polytron PT300 and lysed by three passes
through a microfluidizer at 10,000 psi. The homogenate was
centrifuged at 14,000 g for 60 min at 4.degree. C. and Mes pH 5.0
was added to 50 mM final concentration. The lysate was loaded onto
a 5 ml HiTrap.TM. SP HP (Pharmacia) column previously equilibrated
with buffer A (25 mM sodium phosphate pH 5.5, 10 mM
B-mercaptoethanol) containing 150 mM NaCl. The column was washed
with 4 column volumes (CV) of buffer A, followed by 4 CV of buffer
A containing 150 mM NaCl, and protein was eluted with a 0.3-1.0 M
NaCl gradient in the same buffer. Fractions containing Shh (based
on SDS-PAGE analysis) were pooled, imidazole (pH 7.0) was added to
a final concentration of 20 mM, and the material was loaded onto a
5 ml His Trap (Pharmacia) column equilibrated with buffer B (25 mM
sodium phosphate pH 8.0, 300 mM NaCl, 10 mM s-mercaptoethanol).
After washing with 5 CV of 20 mM imidazole in buffer B and 5 CV of
a 20-50 mM imidazole gradient, the protein was eluted with 5 CV of
250 mM imidazole in the same buffer. Eluted fractions were analyzed
by SDS-PAGE, and the elution pool concentration was determined by
absorbance at 280 nm using an extinction coefficient of 1.17
(g/1).sup.-1 cm.sup.-1 for Shh. The protein was sterilized by 0.2
.mu.M filtration and single-use aliquots stored at -80.degree. C.,
then thawed in cold water immediately prior to use.
Generation of Anti-Shh Rabbit Polyclonal Antibodies
[0202] 250 .mu.g of the his-Shh-N antigen prepared by the procedure
described above was injected into each of two 3-month old New
Zealand white rabbits (numbered 59A and 59B) using 1:1
antigen:complete Freund's Adjuvant, followed by boosting every
other week (with a break between weeks 30 and 41) with 1:1
antigen:incomplete Freund's Adjuvant (Josman labs, LLC). Immune
sera were titered by serial dilution ELISA on the same antigen
(Antibody Solutions, Mountain View, Calif.), with rabbit 59A having
a maximal titer of 1:200 000 at week 9 and rabbit 59B over 1:2 000
000 at week 11. Bleeds from weeks 7-11 of rabbit 59A and weeks 3-11
of rabbit 59B were affinity purified on the Shh-N antigen bound to
CNBr activated Sepharose.RTM. beads and the purified antibodies
dialyzed against PBS and flash-frozen in aliquots for storage at
-20.degree. C. For comparison, the rabbit polyclonal anti-Shh
antibody H-160 (Santa Cruz sc-902, lot# K0104) was raised against
the human Shh aa fragment 41-200.
Generation of Anti-Shh Rabbit Monoclonal Antibodies
[0203] Following boosts at weeks 45 and 52, the spleen of rabbit
59B (with a titer of 1:15 000 at week 46) was excised to rabbit
myeloma cells (240-E1, Epitomics). The hybridoma supernatants were
screened by ELISA on HQ-hShh-N antigen and the positive ones (32)
further screened for reactivity on PFA-fixed hShh-transfected COS
cells by immunofluorescence followed by formalin-fixed,
paraffin-embedded hShh transfected 293 cells by
immunohistochemistry as described below. The 4 chosen clones were
expanded, subcloned by limiting dilution and re-tested as above.
The three best subclones from the single remaining positive parent
(#95) were selected for scale-up in Integra flasks 95.3, 95.7 and
95.9. After 3 months' growth in low-serum media the supernatants
were purified on protein-A Sepharose.RTM., yielding approximately 1
mg 95.3 (Shh:4667), 1.2 mg 95.7 (Shh:4668) and 2 mg 95.9
(Shh:4669).
Immunofluorescence
[0204] COS-7 cells were transiently transfected with untagged
full-length human Shh (Accession NP.sub.--000184), Ihh
(NP.sub.--002172) or Dhh (NP.sub.--066382) in pCMV.Sport6 using
Lipofectamine.TM. 2000 (Invitrogen) reagent according to the
manufacturer's protocol in 8-well LabTekII.TM. slides or 96-well
black walled microscopy plates (Whatman) for screening. After 60 h
transfection, cells were fixed with 3% PFA for 20 min at room
temperature, quenched for 10 min in 50 mM ammonium chloride and
permeabilized with 0.4% saponin (Sigma) in PBS containing 1% BSA
and 2% FBS. Affinity purified rabbit polyclonals and monoclonals
were used at 5 Og/ml, while hybridoma supernatants were diluted
1:2. 5E1 monoclonal anti-Shh (Ericson/Jessell 1998 Cell 87 p661) at
10 g/ml was used as a positive control. Antibody staining was
detected with Cy3-labeled donkey anti-rabbit (or mouse for 5E1)
(Jackson Immunoresearch) and visualized by epifluorescence
microscopy using a Discovery-1 high content screening microscope
(Molecular Devices) and/or a DeltaVision (Applied Precision)
microscope equipped with DAPI, FITC and rhodamine filters.
Immunohistochemistry
[0205] HEK293 cells were transiently transfected with untagged
human Shh full length hShh (block H2006-223(5)), Dhh (H2006-1676)
or hIhh (H2006-1677) in pcDNA3.1 using Lipofectamine.TM. 2000
(Invitrogen) reagent according to the manufacturer's protocol in
ten 15 cm dishes and harvested 48 h later with 5 mM EDTA in PBS.
Cell pellets were processed for formalin fixation and paraffin
embedding according to standard protocols. In brief, the cells were
fixed with 10% Neutral Buffered Formalin, processed in automated
processors, paraffin-embedded (FFPE) and sectioned at 3 .mu.m on
Superfrost.RTM. Plus slides. Slides were then de-paraffinized and
hydrated in water after treatment with a series of xylenes and
alcohols in a Leica.RTM. autostainer and pre-treated for antigen
retrieval with Dako.RTM. TARGET retrieval solution in PT module
(Lab Vision); this method worked better than DakoR High pH and
Triology retrieval methods for 95.9 antibodies. Endogenous
peroxidase activity was then quenched by incubating the slides in
KPL solution (KPL Laboratories) for 4 minutes at room temperature.
After blocking endogenous immunoglobulins with blocking serum,
slides were stained in a three-step protocol on a Dako.RTM.
autostainer, employing various rabbit anti-Hh primary antibodies or
neat hybridoma supernatants, followed by biotinylated anti rabbit
secondaries (Vector Laboratories) then ABC complex (ABC Elite
Kit-Vector Laboratories) and visualized using DAB (Pierce
Laboratories) as a chromogen. To maximize detection of weak
antibodies in supernatants, a further amplification step was added,
using tyramide signal amplification (TSA) followed by
Streptavidin-HRP (Perkin Elmer TSA kit) in place of ABC complex.
Slides were then counterstained with Meyers Hematoxylin and
dehydrated with series of alcohols and xylenes followed by
coverslipping using organic mounting medium (Permamount). Naive
rabbit IgG (Alpha Diagnostics) was used as negative control and
rabbit anti-Hh H-160 (Santa Cruz Biotech Sc-9024) as a positive
control. Staining with purified 95.9 rMab was optimal at 5
.mu.g/ml, and both this and H-160 worked best without TSA
amplification, to minimize background.
Hh Sandwich ELISA Assay.
[0206] ELISA plates were coated overnight at 4.degree. C. with 5
.mu.g/ml anti-Shh/Ihh monoclonal antibody AA.F10 (Curis, Inc) or
95.9 and incubated with increasing concentrations of recombinant
octyl-Shh at room temperature for 1 h in assay diluent (0.5% BSA,
0.05% Tween20, 15 ppm proclin in PBS). After washing, bound
octyl-Shh was detected by sequential incubations of 0.1 .mu.g/ml
biotinylated 5E1 anti-Hh (anti-Shh/Ihh/Dhh) for 1 h, 50 ng/ml
streptavidin-HRP for 30 min, then visualized with TMB substrate and
read at 650 nm. The ELISA was performed in triplicate and OD650 was
plotted vs octyl-Shh concentration.+-.standard deviation of the
mean. Using AA.F10 as the coating Ab, this assay reproducibly
detects octyl-Shh in the range of 78 pg/ml to 10 ng/ml.
Results
Generation of Rabbit Monoclonals to Hh.
[0207] Two rabbit polyclonal antibodies were raised to human Shh-N
expressed in E. coli with an N-terminal unizyme tag. Both rabbits
59A and 59B responded well, with titers of 1:200 000 and over 1:2
million, respectively, by the 11.sup.th week of boosting. All
bleeds with a titer of over 1:7000 were pooled and affinity
purified on the antigen, and the resulting antibody compared with
the current "gold standard" H-160 antibody (Santa Cruz
Biotechnology) by immunofluorescence (IF), immunohistochemistry
(IHC) and Western blotting of Shh-transfected cells. The rabbit
polyclonal stained Shh in the endoplasmic reticulum of stably
transfected Shh-COS cells (FIG. 1A), similar to the staining
obtained with H-160 (FIG. 1C), but with some nuclear background
evident in untransfected COS cells (FIG. 1B). It also recognized
Shh in the cytoplasm (most likely endoplasmic reticulum) and cell
membrane of formalin-5 fixed, paraffinembedded 293 cell pellets by
IHC (FIG. 2C), again with some background staining in untransfected
cells (FIG. 2D), but otherwise slightly stronger than to H-160
staining (FIG. 2B).
[0208] Rabbit monoclonal antibodies (rmAbs) not only provide a
theoretically unlimited supply of reagent, but also have the
advantage of recognizing a more specific epitope with higher
affinity than a mixed polyclonal. The spleen of the higher titer
rabbit 59B was therefore fused with rabbit myeloma cells to
generate rabbit hybridomas. The 96-well supernatants of these were
screened by ELISA on the Shh-N antigen (data not shown) and further
expanded and selected for reactivity by IF and IHC as above. Of 32
initial ELISA+ parental clones, one (clone #95) remained positive
following subcloning and the three strongest IHC+ subclones were
selected: 95.3, 95.7 and 95.9. All three subclones are likely
identical, based on their similar immunostaining of Shh-transfected
cells (FIG. 3 top row) and virtually identical Nterminal sequences
(FIG. 4A), although the presence of endogenous (non-Shh reactive)
heavy chain from the myeloma fusion partner complicated the
sequence analysis.
[0209] Due to its greater yield, 95.9 was selected for scale up and
purification. It recognized Shh in transfected cells by IF (FIG.
1E) and IHC (FIG. 2E), but was cleaner than the polyclonal from
which it was derived, as judged by the much reduced background
staining on untransfected cells by IF (FIG. 1F, 2D). 95.9 rmAb did
not require TSA amplification in order to detect its antigen. The
staining of Shh-transfected cells was specific because another
rabbit monoclonal antibody (R&D systems catalog number 269518)
to an irrelevant antigen only resulted in background cytoplasmic
staining (FIG. 2A and data not shown). Furthermore, the
Shh-specific staining was stronger than that of the H-160 antibody
(compare FIG. 2E with 2B).
[0210] Although raised against Shh-N, 95.9 and its sister clones
95.3 and 95.7 cross-react with the 89% identical Ihh-N and 74%
identical Dhh-N, as judged by IF (FIG. 3A), 1HC (data not shown),
and Western blotting (FIG. 3B) of transfected full-length clones,
similar to H-160, thus we henceforth refer to these antibodies as
anti-Hh. By Western blotting, both the full length Hh (.about.50
kDa) unprocessed ER form of Hh and the cleaved Hh-N (.about.25 kDa)
were detected in transfected cells (although in stable Shh/COS
cells where there is less total Shh expression, the full length
band is less obvious or absent (data not shown). The .about.95 kDa
band detected by H-160, which is thought to be non-specific, due to
its size and presence in untransfected cells, was not recognized in
untransfected cells by 95.9 (although it did appear in the
Dhh-transfected cells, where it is presumably some kind of Dhh
dimer). All three anti-Hh clones and H-160 also cross-reacted with
mouse Shh-N using all three techniques (data not shown), as
expected since there is only one amino acid difference (Ser 67 of
the human sequence is Thr in mouse), allowing us to stain mouse
embryos as one means of validating the specificity and sensitivity
of 95.9.
[0211] The epitope of 95.9 was mapped by tryptic digestion of
his-tagged hShh to residues 75-96, which are 100% identical in
hIhh, and are only 3 aa different in hDhh (FIG. 5) thus suggesting
that 95.9 will detect Shh and Ihh with similar, if not identical
sensitivity, as well as explaining the ability of the antibody to
cross-react with all three ligands. Additionally, this peptide is
found on the opposite face of Shh to the previously established Ptc
and 5E1 blocking antibody binding sites {Pepinsky, 2000 #4}, which
supports the inability of 95.9 to compete with 5E1 for Hh binding
(data not shown, but see FIG. 11.
95.9 Anti-Hh rmAb Shows Greater Sensitivity and Specificity than
the H-160 Polyclonal by IHC Staining
[0212] Several anti-Hh antibodies have been shown to recognize
recombinant Hh, but staining of the lower levels present in
endogenous tissues or tumors has been less successful, especially
in FFPE specimens (refs). Since 95.9 was selected for its ability
to recognize FFPE-fixed Hh, it was of interest to determine if it
was sensitive and specific enough to detect endogenous Hh. To this
end, we examined developing mouse embryos, since the distribution
of Shh mRNA during development has been well documented by in situ
hybridization (ISH) to be abundant at the E10.5 stage in the
notochord and floorplate of the ventral neural tube from where Shh
is thought to diffuse and create a morphogen gradient to specify
the fate of the various neurons in the neural tube (refs); as well
as in the mid-gut, where it is implicated in the morphogenesis of
the gut epithelium {Bitgood, 1995 #1} and in hind limb buds, where
it regulates proper digit formation {Chuong, 2000 #2; Johnson, 1994
#3}. Direct evidence that the mRNA is translated comes from
notochord and floorplate staining of frozen embryos with the
monoclonal anti-Shh antibody 5E1 (ref), but this antibody
insufficiently stains shh in FFPE fixed specimens such as routine
tumor biopsies, thus limiting its potential as a diagnostic for
putative Hh-expressing tumors. In contrast, the anti-Shh antibody
95.9 specifically stained the notochord and ventral floor plate of
E10.5 mouse embryos (brown stain in FIG. 6A), much more strongly
than the H-160 antibody (FIG. 6B) under the same staining
conditions (which were previously found also to be optimal for
H-160 on Shh/293 cell pellets). At E11.5, not only the ventral
neural tube and floorplate were obvious (FIG. 6C), but also faint
staining of structures extending away from the floorplate (FIG.
6D). While this could be consistent with the postulated role of Hh
as morphogen, this should be confirmed by ISH to confirm any
overlap with Gli1 or Ptc1 to determine if this expression is real,
as Shh mRNA would not overlap in the receiving cells)). If real, it
would be suggestive of the first direct visualization of Hh
gradient. Hh staining was also present in the neuroepithelium
developing brain between the telecephalic vesicle and 4th ventricle
(E11.5; FIG. 6E) as well as the mid-gut epithelium (FIG. 6F), as
expected from earlier ISH results. There was no staining in any
parts of the embryo where Hh was not expected to be found,
illustrating the specificity of 95.9.
[0213] As a further test for 95.9 sensitivity, we analyzed by IHC a
panel of 36 human colon cancer cell lines in which we had
pre-determined mRNA levels for Shh (and Ihh and Dhh) by Q-PCR. The
critical threshold (Ct) values for Shh ranged from undetectable in
RKO and Colo320 cells (below a Ct of 38) to 24 for the highest
expressing cell lines. Importantly, none of the cell lines that
lacked all three Hh mRNAs (such as DLD-1) showed any staining with
95.9, thereby demonstrating specificity of the antibody to
hedgehog. Based on cytoplasmic and membranous staining of Hh by
95.9, various cell lines could be grouped into three positive
categories: low (scored as 1+; e.g. FIG. 7B); moderate (2+; e.g.
FIG. 7C) or high (3+; e.g. FIG. 7D) Hh-expressing. The .DELTA.Ct
values following normalization to the housekeeping gene
.beta.-glucuronidase (GUSB) were plotted versus IHC scores,
illustrating a trend towards higher IHC scores with higher mRNA
values (lower .DELTA.Ct values), with a Spearman coefficient of -5
0.61 (FIG. 7E). The correlation was not perfect because some of the
cell lines also express Ihh, which is likely equally well
recognized by 95.9 (based on 100% sequence identity in the epitope;
FIG. 5), in addition to Shh (notably the 3+cell line SW403 and the
2+cell line HT29), but nonetheless is statistically significant
(p=0.0001). These data suggest that 95.9 can usefully identify
Hh-expressing FFPE-processed cancer cell lines by IHC. Importantly,
two of the colorectal xenograft cell lines that respond well to
GDC-0449 (and 5E1) in vivo, LS180 and HT55 (data not shown),
express Hh at only 1+levels (FIG. 7E), suggesting that it may be
the presence of Hh per se, rather than high expression of Hh that
is important in tumor selection for Hh antagonist therapy. In this
respect, the higher sensitivity of 95.9 than H160 may allow it to
detect a higher proportion of 1+expressors and therefore makes it a
better diagnostic antibody.
[0214] Detection of Hh in primary human tumor biopsies could be
useful for selection of type III (paracrine Hh-ligand-driven)
cancer patients for therapy with Hh antagonists, since tumors that
do not express Hh would be unlikely to respond such therapy. We
therefore sought to determine if 95.9 was capable of detecting Hh
in a human ovarian tumor that had previously been shown to express
Shh by ISH. Indeed, 95.9 gave robust labeling of the Shh-(+) but
not a different Shh-(-) ovarian tumor (FIG. 8), confirming its
utility and specificity.
[0215] Encouraged by the specificity and apparently superior
sensitivity of 95.9 to H-160, we proceeded to determine the
prevalence of Hh expression in a larger array of specimens from
ovary (72 tumors and 2 normal, data not shown), colon (99 tumors
and 44 normal) and pancreas (105 tumors and 17 normal) and
classified the cytoplasmic staining as absent (0+), weak (1+),
moderate (2+) or strong (3+), with representative images from each
category for each tissue type being shown in FIG. 9A. Approximately
20% of both tumor and normal colon samples (FIG. 9B) lacked
specific cytoplasmic and membranous Hh staining (any nuclear
background was considered non-specific, since Hh is not expected to
be present in this part of the cell). The remaining 80% were
Hh-positive, with mostly 1+expression (68% tumors and 54% normal),
fewer with 2+ expression (10% tumor and 23% normal) and even fewer
with strong 3+ staining (3% tumor and no normal). Similarly, low
expression was found in most of the ovarian samples (57% tumor and
2/2 normal were considered 1+), with 31% in the 2+and 6% in the
3+categories and 7% negative tumors. Likewise, 23% of 105
pancreatic tumors were considered negative, compared to 41% of
normal pancreas specimens; 70% of tumors and 41% normal expressed
low (1+) levels of Hh; 7% of tumors and 18% of normals expressed
2+levels, and no specimens displayed strong staining. These results
indicate that while Hh expression does not appear to be much
stronger in tumors than normal tissues, there is a range of
expression among specimens. Importantly, up to 23% tumor samples
lacked Hh expression in this analysis, suggesting that it may be
worthwhile to evaluate Hh expression in patients undergoing
clinical trials with Hh antagonists to determine if any lack of
response correlates with lack of Hh expression. It is also possible
that that anti-Hh antibodies of the invention can be used to
determined if Hh expression level correlates with response to
antagonist treatment.
[0216] Finally, we wanted to determine the sensitivity of the
anti-Hh antibody 95.9 in a low expressing tissue, such as hair
follicles. Hh mRNA has been detected by in situ hybridization in
hair follicles {Iseki, 1996 #8} and furthermore, injection of the
blocking antibody 5E1 or the Hh antagonist cyclopamine prevents
hair development in mouse embryos {Wang, 2000 #6}; {Chiang, 1999
#10}, thus strongly suggesting that Hh signaling in this organ is
essential. However, Hh protein has not been demonstrated in the
hairs of FFPE skin specimens by IHC, most likely due inadequate
antibody sensitivity. Hh signaling is known to be elevated during
the anagen phase of hair growth, with less activity during telogen
{Sato, 1999 #9} and in adult mice, hair growth is synchronized such
that anagen occurs at 4 weeks of age and showed a robust signal at
this stage of growth for both Shh protein (FIG. 10A), as detected
by 95.9 staining. In humans, hair growth is not synchronized, but a
subset of follicles should be in anagen phase at any given time.
Indeed, we observed approximately 10% hair follicles from human
fetal scalp showing Shh-specific signal using an antisense (FIG.
10B) but not a sense probe (Data not shown). Accordingly, a subset
of hair follicles showed 95.9-reactivity, with well-defined
staining in the proximal epithelium above the dermal papilla (FIG.
10 C,E), much stronger than the very faint staining obtained with
H160 (FIG. 10D, F). Thus, 95.9 is sensitive enough to detect low
levels of Hh in anagen hair follicles and is superior to the H160
antibody in this respect. This suggests that 95.9 is likely to
detect a greater proportion of Hh-expressing tumor samples than
H160 and so is the reagent of choice for development of an IHC kit
for clinical selection of prospective patients for Hh-antagonist
therapy.
Example 2
Microarray Analysis to Detect Downregulation of Hedgehog
Polypeptides in Cancer or Tumors
[0217] Nucleic acid microarrays, often containing thousands of gene
sequences, are useful for identifying differentially expressed
genes in diseased tissues as compared to their normal counterparts.
Using nucleic acid microarrays, test and control mRNA samples from
test and control tissue samples are reverse transcribed and labeled
to generate cDNA probes. The cDNA probes are then hybridized to an
array of nucleic acids immobilized on a solid support. The array is
configured such that the sequence and position of each member of
the array is known. For example, a selection of genes known to be
expressed in certain disease states may be arrayed on a solid
support. Hybridization of a labeled probe with a particular array
member indicates that the sample from which the probe was derived
expresses that gene. If the hybridization signal of a probe from a
test (disease tissue) sample is greater than hybridization signal
of a probe from a control (normal tissue) sample, the gene or genes
overexpressed in the disease tissue are identified. The implication
of this result is that an overexpressed protein in a diseased
tissue is useful not only as a diagnostic marker for the presence
of the disease condition, but also as a therapeutic target for
treatment of the disease condition.
[0218] The methodology of hybridization of nucleic acids and
microarray technology is well known in the art. In the present
example, the specific preparation of nucleic acids for
hybridization and probes, slides, and hybridization conditions are
all detailed in PCT Patent Application Serial No. PCT/US01/10482,
filed on Mar. 30, 2001 and which is herein incorporated by
reference.
Example 3
Quantitative Analysis of Hedgehog mRNA Expression
[0219] In this assay, a 5' nuclease assay (for example, TaqMan) and
real-time quantitative PCR (for example, ABI Prism 7700.RTM.
Sequence Detection System (Perkin Elmer, Applied Biosystems
Division, Foster City, Calif.)), is used to find genes that are
significantly overexpressed in a cancerous glioma tumor or tumors
as compared to other cancerous tumors or normal non-cancerous
tissue. The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor gene expression in real time. Two
oligonucleotide primers (whose sequences are based upon the gene or
EST sequence of interest) are used to generate an amplicon typical
of a PCR reaction. A third oligonucleotide, or probe, is designed
to detect nucleotide sequence located between the two PCR primers.
The probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the PCR amplification reaction,
the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments
disassociate in solution, and signal from the released reporter dye
is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated for each new molecule
synthesized, and detection of the unquenched reporter dye provides
the basis for quantitative and quantitative interpretation of the
data. This assay is well known and routinely used in the art to
quantitatively identify gene expression differences between two
different human tissue samples, see, e.g., Higuchi et al.,
Biotechnology 10:413-417 (1992); Livak et al., PCR Methods Appl.,
4:357-362 (1995); Heid et al., Genome Res. 6:986-994 (1996);
Pennica et al., Proc. Natl. Acad. Sci. USA 95(25):14717-14722
(1998); Pitti et al., Nature 396(6712):699-703 (1998) and Bieche et
al., Int. J. Cancer 78:661-666 (1998).
[0220] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism.RTM. 7700 Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0221] The starting material for the screen is mRNA isolated from a
variety of different cancerous tissues. The mRNA is quantitated
precisely, e.g., fluorometrically. As a negative control, RNA is
isolated from various normal tissues of the same tissue type as the
cancerous tissues being tested. Frequently, tumor sample(s) are
directly compared to "matched" normal sample(s) of the same tissue
type, meaning that the tumor and normal sample(s) are obtained from
the same individual.
[0222] 5' nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a 5 particular target
sequence in a nucleic acid sample when comparing cancer mRNA
results to normal human mRNA results. As one Ct unit corresponds to
1 PCR cycle or approximately a 2-fold relative increase relative to
normal, two units corresponds to a 4-fold relative increase, 3
units corresponds to an 8-fold relative increase and so on, one can
quantitatively and quantitatively measure the relative fold
increase in mRNA expression between two or more different tissues.
In this regard, it is well accepted in the art that this assay is
sufficiently technically sensitive to reproducibly detect an at
least 2-fold increase in mRNA expression in a human tumor sample
relative to a normal control.
Example 4
In Situ Hybridization
[0223] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[0224] In situ hybridization is performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1:169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues are sectioned,
deparaffinized, deproteinated in proteinase K (20 g/ml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-P]
UTP-labeled antisense riboprobe are generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides are dipped in
Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
.sup.33P-Riboprobe Synthesis
[0225] 6.0 .mu.l (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000
Ci/mmol) were speed vac dried. To each tube containing dried
.sup.33P-UTP, the following ingredients were added:
[0226] 2.0 .mu.l 5.times. transcription buffer
[0227] 1.0 .mu.l DTT (100 mM)
[0228] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H2O)
[0229] 1.0 .mu.l UTP (50 .mu.M)
[0230] 1.0 .mu.l Rnasin
[0231] 1.0 .mu.l DNA template (1 .mu.g)
[0232] 1.0 .mu.l H.sub.2O
[0233] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[0234] The tubes are incubated at 37.degree. C. for one hour. 1.0
.mu.l RQ1 DNase is added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
are added, and the mixture was pipetted onto DE81 paper. The
remaining solution is loaded in a Microcon-50 ultrafiltration unit,
and spun using program 10 (6 minutes). The filtration unit is
inverted over a second tube and spun using program 2 (3 minutes).
After the final recovery spin, 100 .mu.l TE is added. 1 .mu.l of
the final product is pipetted on DE81 paper and counted in 6 ml of
Biofluor II.
[0235] The probe is run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III is added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
probe is immediately placed on ice. The wells of gel are flushed,
the sample loaded, and run at 180-250 volts for 45 minutes. The gel
is wrapped in saran wrap and exposed to XAR film with an
intensifying screen in -70.degree. C. freezer one hour to
overnight.
.sup.33P-Hybridization
[0236] A. Pretreatment of Frozen Sections
[0237] The slides are removed from the freezer, placed on aluminum
trays and thawed at room temperature for 5 minutes. The trays are
placed in 55.degree. C. incubator for five minutes to reduce
condensation. The slides are fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the
sections are washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections are dehydrated in 70%, 95%, 100% ethanol,
2 minutes each.
[0238] B. Pretreatment of Paraffin-Embedded Sections
[0239] The slides are deparaffinized, placed in SQ H2O, and rinsed
twice in 2.times.SSC at room temperature, for 5 minutes each time.
The sections are deproteinated in 20 .mu.g/ml proteinase K (500
.mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer; 37.degree. C.,
15 minutes)--human embryo, or 8.times. proteinase K (100 .mu.l in
250 ml Rnase buffer, 37.degree. C., 30 minutes)--formalin tissues.
Subsequent rinsing in 0.5.times.SSC and dehydration are performed
as described above.
[0240] C. Prehebridization
[0241] The slides are laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)-saturated filter paper.
[0242] D. Hybridization
[0243] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide are heated at 95.degree. C. for 3 minutes. The
slides are cooled on ice, and 48 .mu.l hybridization buffer are
added per slide. After vortexing, 50 .mu.l .sup.33P mix are added
to 50 .mu.l prehybridization on slide. The slides are incubated
overnight at 55.degree. C.
[0244] E. Washes
[0245] Washing is done 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA, V.sub.t=4
L), followed by RNaseA treatment at 37.degree. C. for 30 minutes
(500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml). The
slides are washed 2.times.10 minutes with 2.times.SSC, EDTA at room
temperature. The stringency wash conditions can be as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
[0246] F. Oligonucleotides
[0247] In situ analysis is performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
is obtained so as to be complementary to the nucleic acids (or the
complements thereof) as shown in the accompanying figures.
Example 5
Expression of Anti-Hh Antibody in E. coli
[0248] This example illustrates preparation of an unglycosylated
form of anti-hedgehog antibody by recombinant expression in E.
coli.
[0249] The DNA sequence encoding the preceding antibody sequences
is initially amplified using selected PCR primers. The primers
should contain restriction enzyme sites which correspond to the
restriction enzyme sites on the selected expression vector. A
variety of expression vectors may be employed. An example of a
suitable vector is pBR322 (derived from E. coli; see Bolivar et
al., Gene, 2:95 (1977)) which contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction
enzyme and dephosphorylated. The PCR amplified sequences are then
ligated into the vector. The vector will preferably include
sequences which encode for an antibiotic resistance gene, a trp
promoter, a polyhis leader (including the first six STII codons,
polyhis sequence, and enterokinase cleavage site), the anti-hedghog
antibody coding region, lambda transcriptional terminator, and an
argil gene.
[0250] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0251] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0252] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized heavy and light chains of the
anti-hedgehog antibody can then be purified using a metal chelating
column under conditions that allow tight binding of the
protein.
[0253] The preceding heavy and light chain polypeptide sequences
may be expressed in E. coli in a poly-His tagged form, using the
following procedure. The DNA encoding heavy and light chains of the
antihedgehog antibody is initially amplified using selected PCR
primers. The primers will contain restriction enzyme sites which
correspond to the restriction enzyme sites on the selected
expression vector, and other useful sequences providing for
efficient and reliable translation initiation, rapid purification
on a metal chelation column, and proteolytic removal with
enterokinase. The PCR-amplified, poly-His tagged sequences are then
ligated into an expression vector, which is used to transform an E.
coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[0254] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 10 M guanidine, 20 mM Tris,
pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added
to make final concentrations of 0.1M and 0.02 M, respectively, and
the solution is stirred overnight at 4.degree. C. This step results
in a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0255] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a
Poros.RTM. R1/H reversed phase column using a mobile buffer of 0.1%
TFA with elution with a gradient of acetonitrile from 10 to 80%.
Aliquots of fractions with A280 absorbance are analyzed on SDS
polyacrylamide gels and fractions containing homogeneous refolded
protein are pooled. Generally, the properly refolded species of
most proteins are eluted at the lowest concentrations of
acetonitrile since those species are the most compact with their
hydrophobic interiors shielded from interaction with the reversed
phase resin. Aggregated species are usually eluted at higher
acetonitrile concentrations. In addition to resolving misfolded
forms of proteins from the desired form, the reversed phase step
also removes endotoxin from the samples.
[0256] Fractions containing the desired folded protein are pooled
and the acetonitrile removed using a gentle stream of nitrogen
directed at the solution. Proteins are formulated into 20 mM Hepes,
pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or
by gel filtration using G25 Superfine (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
Example 6
Expression of Anti-Hedgehog Antibody 5 in Mammalian Cells
[0257] This example illustrates preparation of a potentially
glycosylated form of anti-hedgehog antibody by recombinant
expression in mammalian cells.
[0258] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
may be employed as the expression vector. Optionally, DNA encoding
the heavy and light chains of the anti-hedgehog antibody described
herein is ligated into pRK5 with selected restriction enzymes to
allow insertion of such DNA using ligation methods such as
described in Sambrook et al., supra. The resulting vector is called
anti-Hh-DNA.
[0259] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-anti-Hh DNA is mixed with about 1 .mu.g DNA encoding
the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and
dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl2. To this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES
(pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is
allowed to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0260] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml.sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of the heavy and light chains of the anti-hedgehog
antibody. The cultures containing transfected cells may undergo
further incubation (in serum free medium) and the medium is tested
in selected bioassays.
[0261] In another embodiment, the anti-hedgehog antibody can be
expressed in CHO cells. The pRK5-anti-Hh can be transfected into
CHO cells using known reagents such as CaPO.sup.4 or DEAE-dextran.
As described above, the cell cultures can be incubated, and the
medium replaced with culture medium (alone) or medium containing a
radiolabel such as .sup.35S-methionine. After determining the
presence of the anti-Hh antibody, the culture medium may be
replaced with serum free medium. Preferably, the cultures are
incubated for about 6 days, and then the conditioned medium is
harvested. The medium containing the expressed anti-Hh antibody can
then be concentrated and purified by any selected method.
[0262] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0263] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5= and
3= of the DNA of interest to allow the convenient shuttling of
cDNA=S. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0264] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents SUPERFECT.RTM. (Quiagen),
DOSPER.RTM. or FUGENE.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.7 cells are frozen in an ampule for further growth
and production as described below.
[0265] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH ie
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 Fm filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0266] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0267] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Example 7
Purification of Anti-Hh Antibodies Using Anti-Hh Specific
Antibodies
[0268] Native or recombinant anti-Hh antibodies may be purified by
a variety of standard techniques in the art of protein
purification. For example, pro-, mature or pre-polypeptide variants
of the preceding heavy and light chain sequences are purified by
immunoaffinity chromatography using antibodies specific for such
sequences. In general, an immunoaffinity column is constructed by
covalently coupling the respective heavy and light chains of the
anti-Hh antibody to an activated chromatographic resin.
[0269] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated Sepharose.RTM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0270] Such an immunoaffinity column is utilized in the
purification of the preceding heavy and light chain sequences by
preparing a fraction from cells containing such sequences in a
soluble form. This preparation is derived by solubilization of the
whole cell or of a subcellular fraction obtained via differential
centrifugation by the addition of detergent or by other methods
well known in the art. Alternatively, soluble heavy and light chain
polypeptide containing a signal sequence may be secreted in useful
quantity into the medium in which the cells are grown.
[0271] A soluble heavy and light chain preparations are passed over
the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of such sequences
(e.g., high ionic strength buffers in the presence of detergent).
Then, the column is eluted under conditions that disrupt the
binding between the antibody/substrate (e.g., a low pH buffer such
as approximately pH 2-3, or a high concentration of a chaotrope
such as urea or thiocyanate ion), and the heavy and/or light chain
polypeptide, respectively, is collected.
Example 7
Immunohistochemistry Using Anti-Hh Antibodies
[0272] Experimental Design:
[0273] We examined Hh ligand protein expression in colorectal and
ovarian carcinomas using 1 ug/ml and 5 ug/ml of 95.9 antibody.
Twenty colorectal and 20 ovarian carcinomas were stained with 1
ug/ml and 5 ug/ml of the 95.9 anti-Hh antibody using a previously
established protocol. Both a numeric and an alphanumeric scoring
system were employed to capture the amount of tumor epithelial
staining. The numeric scoring system estimates the total level of
staining for each specimen (0=no staining, 1=weak, 2=moderate,
3=strong). The alphanumeric score captures 1) the % of tumor
epithelium demonstrating any level of staining (0=no staining,
1=<25%, 2=25-75%, 3=>75%) and 2) the predominant intensity of
staining (A=weak, B=moderate, C=strong). Tumors demonstrating less
than 5% of tumor epithelium staining received a score of 0. The
results are shown in Table 2. We found that a greater range of
staining intensity was observed with 5 ug/ml primary antibody. We
also found staining of Shh in neural tube cells with 95.5 and
ovarian cancer cells, but not in normal ovarian tissue. Thus mAb
95.5 is a useful and sensitive antibody for diagnostic use in
cancer, particularly in immunohistochemical staining
TABLE-US-00003 TABLE 2 Histologic Findings Colon Tumors Ovary
Tumors Sample IHC IHC Sample IHC IHC No. (lug/ml) (5 ug/ml) Name
(lug/ml) (5 ug/ml) 1 1(1A) 1(3A) 21 0 1(1B) 2 1(3A) 2(3A) 22 1(2A)
2(2B) punctate 3 0 1(2A) 23 0 1(2A) 4 1(2A) 2(3B) 24 1(1A) 1 (1B) 5
0 0 25 0 1(3A) 6 1(2A) 2(3B) 26 0 1(2A) punctate 7 0 1(3A) 27 2(2B)
2(2C) 8 0 1(2A) 28 1(2A) 2(2B) punctate 9 0 1(2A) 29 0 1(3A) 10 0
1(3A) 30 0 1(2A) (focally (focally strong) strong) 11 0 1(3A) 31 0
1(2A) 12 0 0 32 0 1(2A) 13 0 2(3A) 33 0 1(2A) 14 0 1(2A) 34 0 1(1A)
15 1(1B) 2(2B) 35 0 1(3A) Punctate stromal 16 0 0 36 0 0 17 0 1(2A)
37 0 1(2A) 18 0 1(2A) 38 0 1(2A) 19 1(3A) 2(3B) 39 0 1(1A) 20 0
1(2A) 40 0 1(3A)
Sequence CWU 1
1
18124PRTMus musculus 1Gln Ser Val Lys Glu Ser Gly Gly Gly Leu Val
Gln Pro Glu Gly 1 5 10 15Ser Leu Thr Leu Thr Cys Thr Val Ser 20
210PRTMus musculus 2Gly Phe Ser Leu Ser Ser Tyr Asp Met Ser 5
10313PRTMus musculus 3Trp Val Arg Gln Ala Pro Gly Ser Gly Leu Glu
Trp Ile 5 10 417PRTMus musculus 4Gly Gly Ile Leu Ser Gly Gly Ser
Ala Tyr Tyr Ala Ser Trp Ala 1 5 10 15Lys Ser530PRTMus musculus 5Arg
Ser Thr Ile Thr Lys Asn Thr Asn Leu Asn Thr Val Thr Leu 1 5 10
15Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 20 25
30613PRTMus musculus 6Ala Arg Gly Ile Tyr Pro Val Gly Thr Asn Tyr
Asn Ile 5 10 712PRTMus musculus 7Trp Gly Pro Gly Thr Leu Val Thr
Val Ser Ser Gly 5 10 8322PRTMus musculus 8Gln Pro Lys Ala Pro Ser
Val Phe Pro Leu Ala Pro Cys Cys Gly 1 5 10 15Asp Thr Pro Ser Ser
Thr Val Thr Leu Gly Cys Leu Val Lys Gly 20 25 30Tyr Leu Pro Glu Pro
Val Thr Val Thr Trp Asn Ser Gly Thr Leu 35 40 45Thr Asn Gly Val Arg
Thr Phe Pro Ser Val Arg Gln Ser Ser Gly 50 55 60Leu Tyr Ser Leu Ser
Ser Val Val Ser Val Thr Ser Ser Ser Gln 65 70 75Pro Val Thr Cys Asn
Val Ala His Pro Ala Thr Asn Thr Lys Val 80 85 90Asp Lys Thr Val Ala
Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro 95 100 105Pro Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 110 115 120Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 125 130 135Cys Val
Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe 140 145 150Thr
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro 155 160
165Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr 170
175 180Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys
185 190 195Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 200 205 210Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val
Tyr Thr 215 220 225Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser
Val Ser Leu 230 235 240Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp
Ile Ser Val Glu 245 250 255Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn
Tyr Lys Thr Thr Pro 260 265 270Ala Val Leu Asp Ser Asp Gly Ser Tyr
Phe Leu Tyr Ser Lys Leu 275 280 285Ser Val Pro Thr Ser Glu Trp Gln
Arg Gly Asp Val Phe Thr Cys 290 295 300Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 305 310 315Ile Ser Arg Ser Pro Gly
Lys 320 924PRTMus musculus 9Asp Ile Ala Val Leu Thr Gln Thr Pro Ser
Pro Val Ser Ala Ala 1 5 10 15Val Gly Gly Thr Val Thr Ile Asn Cys 20
1012PRTMus musculus 10Gln Ser Ser Pro Ser Val Tyr Ser Asn Tyr Leu
Ala 5 10 1114PRTMus musculus 11Trp Tyr Gln Gln Lys Pro Gly Gln Pro
Pro Lys Leu Leu Ile 5 10 128PRTMus musculus 12Tyr Tyr Ala Ser Thr
Leu Ala Ser 5 1332PRTMus musculus 13Gly Val Pro Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Glu Phe 1 5 10 15Thr Leu Thr Ile Ser Asp Leu
Glu Cys Ala Asp Ala Ala Thr Tyr 20 25 30Tyr Cys1411PRTMus musculus
14Ala Gly Gly Tyr Ile Asp Thr Ser Asp Thr Ala 5 10 15114PRTMus
musculus 15Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp Pro Val
Ala 1 5 10 15Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val
Ala Thr 20 25 30Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe
Pro Asp 35 40 45Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr
Thr Gly 50 55 60Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys
Thr Tyr 65 70 75Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr
Asn Ser 80 85 90His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr
Ser Val 95 100 105Val Gln Ser Phe Asn Arg Gly Asp Cys 110
16174PRTHomo sapiens 16Cys Gly Pro Gly Arg Gly Phe Gly Lys Arg Arg
His Pro Lys Lys 1 5 10 15Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile
Pro Asn Val Ala Glu 20 25 30Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu
Gly Lys Ile Ser Arg 35 40 45Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro
Asn Tyr Asn Pro Asp 50 55 60Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly
Ala Asp Arg Leu Met 65 70 75Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala
Leu Ala Ile Ser Val 80 85 90Met Asn Gln Trp Pro Gly Val Lys Leu Arg
Val Thr Glu Gly Trp 95 100 105Asp Glu Asp Gly His His Ser Glu Glu
Ser Leu His Tyr Glu Gly 110 115 120Arg Ala Val Asp Ile Thr Thr Ser
Asp Arg Asp Arg Ser Lys Tyr 125 130 135Gly Met Leu Ala Arg Leu Ala
Val Glu Ala Gly Phe Asp Trp Val 140 145 150Tyr Tyr Glu Ser Lys Ala
His Ile His Cys Ser Val Lys Ala Glu 155 160 165Asn Ser Val Ala Ala
Lys Ser Gly Gly 170 17175PRTHomo sapiens 17Cys Gly Pro Gly Arg Val
Val Gly Ser Arg Arg Arg Pro Pro Arg 1 5 10 15Lys Leu Val Pro Leu
Ala Tyr Lys Gln Phe Ser Pro Asn Val Pro 20 25 30Glu Lys Thr Leu Gly
Ala Ser Gly Arg Tyr Glu Gly Lys Ile Ala 35 40 45Arg Ser Ser Glu Arg
Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pro 50 55 60Asp Ile Ile Phe Lys
Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu 65 70 75Met Thr Gln Arg Cys
Lys Asp Arg Leu Asn Ser Leu Ala Ile Ser 80 85 90Val Met Asn Gln Trp
Pro Gly Val Lys Leu Arg Val Thr Glu Gly 95 100 105Trp Asp Glu Asp
Gly His His Ser Glu Glu Ser Leu His Tyr Glu 110 115 120Gly Arg Ala
Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Asn Lys 125 130 135Tyr Gly
Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp 140 145 150Val
Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser Val Lys Ser 155 160
165Glu His Ser Ala Ala Ala Lys Thr Gly Gly 170 17518176PRTHomo
sapiens 18Cys Gly Pro Gly Arg Gly Pro Val Gly Arg Arg Arg Tyr Ala
Arg 1 5 10 15Lys Gln Leu Val Pro Leu Leu Tyr Lys Gln Phe Val Pro
Gly Val 20 25 30Pro Glu Arg Thr Leu Gly Ala Ser Gly Pro Ala Glu Gly
Arg Val 35 40 45Ala Arg Gly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn
Tyr Asn 50 55 60Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Ser Gly Ala
Asp Arg 65 70 75Leu Met Thr Glu Arg Cys Lys Glu Arg Val Asn Ala Leu
Ala Ile 80 85 90Ala Val Met Asn Met Trp Pro Gly Val Arg Leu Arg Val
Thr Glu 95 100 105Gly Trp Asp Glu Asp Gly His His Ala Gln Asp Ser
Leu His Tyr 110 115 120Glu Gly Arg Ala Leu Asp Ile Thr Thr Ser Asp
Arg Asp Arg Asn 125 130 135Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val
Glu Ala Gly Phe Asp 140 145 150Trp Val Tyr Tyr Glu Ser Arg Asn His
Val His Val Ser Val Lys 155 160 165Ala Asp Asn Ser Leu Ala Val Arg
Ala Gly Gly 170 175
* * * * *