U.S. patent application number 15/677795 was filed with the patent office on 2018-07-26 for antibodies specific to heterodimers of bcl-2 family and uses thereof.
The applicant listed for this patent is EUTROPICS PHARMACEUTICALS, INC.. Invention is credited to Michael H. CARDONE.
Application Number | 20180208672 15/677795 |
Document ID | / |
Family ID | 41264999 |
Filed Date | 2018-07-26 |
United States Patent
Application |
20180208672 |
Kind Code |
A1 |
CARDONE; Michael H. |
July 26, 2018 |
ANTIBODIES SPECIFIC TO HETERODIMERS OF BCL-2 FAMILY AND USES
THEREOF
Abstract
Isolated antibodies specifically binding to heterodimers of the
Bcl-2 family and uses thereof for detecting presence of Bcl-2
heterodimers in a patient.
Inventors: |
CARDONE; Michael H.;
(Dorchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EUTROPICS PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Family ID: |
41264999 |
Appl. No.: |
15/677795 |
Filed: |
August 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14266424 |
Apr 30, 2014 |
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15677795 |
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13428889 |
Mar 23, 2012 |
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14266424 |
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12437127 |
May 7, 2009 |
8168755 |
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13428889 |
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61051206 |
May 7, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57492 20130101;
C07K 16/18 20130101; C07K 2317/622 20130101; C07K 16/30
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; G01N 33/574 20060101 G01N033/574; C07K 16/18 20060101
C07K016/18 |
Claims
1-9. (canceled)
10. A method for detecting the presence of a heterodimer of the
Bcl-2 family in a fixed tissue sample, comprising (a) contacting an
isolated antibody specifically binding to a heterodimer selected
from Bim and Mcl-1, Bim and Bcl-2, and Bid and Bcl-2 to a fixed
tissue sample suspected of having a heterodimer of the Bcl-2
family; (b) detecting a signal that indicates binding of the
antibody to the heterodimer, and (c) determining the presence of
the heterodimer in the fixed tissue sample based on the intensity
of the signal.
11-12. (canceled)
13. The method of claim 10, wherein the fixed tissue sample is a
peripheral blood sample, a lymph-node sample, a bone marrow sample,
or an organ tissue sample.
14. The method of claim 10, wherein the fixed tissue sample is a
mitochondrial fraction.
15-21. (canceled)
22. The method of claim 10, wherein the fixed tissue sample is a
paraffin embedded tissue sample.
23. The method of claim 10, wherein the fixed tissue sample is a
frozen thin section tissue sample.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. provisional Patent
Application No. 61/051,206, filed May 7, 2008, the content of which
is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Apoptosis is a process of programmed cell death mediated by
a number of signaling pathways that converge at the mitochondria. A
group of mitochondrial proteins, i.e., the Bcl-2 proteins,
regulates this process. More specifically, pro-apoptotic and
anti-apoptotic Bcl-2 proteins form heterodimers with their cognate
regulating Bcl-2 proteins (i.e., the BH3-only Bcl-2 proteins),
thereby executing cell death or survival signals.
[0003] Essentially all effective cancer dugs induce apoptosis in
target cancer cells. However, different cancer cells respond to an
apoptosis-inducing drug in different manners. This is due to the
presence of different heterodimers between the pro/anti-apoptotic
Bcl-2 proteins and the regulatory BH3-only Bcl-2 proteins in those
cancer cells. Determining the presence of these heterodimers in a
cancer patient helps assessing that patient's responsiveness to an
apoptosis-inducing cancer drug.
SUMMARY OF THE INVENTION
[0004] One aspect of this invention features an isolated antibody
that specifically binds to a heterodimer of the Bcl-2 family (i.e.,
a Bcl-2 heterodimer). The Bcl-2 family includes both Bcl-2 proteins
(monomers) and naturally-occurring heterodimers formed between two
Bcl-2 proteins. The heterodimer contains a first Bcl-2 protein
(e.g., Bim, Bid, Bad, Puma, Noxa, Bak, Hrk, Bax, or Mule) and a
second Bcl-2 protein (e.g., Mcl-1, Bcl-2, Bel-XL, Bfl-1 or
Bcl-w).
[0005] The antibody of this invention can be a monoclonal antibody,
a polyclonal antibody, a chimeric antibody, or a humanized
antibody. It also can be a functional fragment of a whole antibody,
such as F(ab').sub.2, Fab', F(ab).sub.2), and Fab. Alternatively,
the antibody of this invention is a single chain antibody, e.g., a
single chain variable fragment (scFv).
[0006] Another aspect of this invention is a method for detecting
the presence of a heterodimer of the Bcl-2 family using any of the
antibodies described above. This method includes (i) providing a
tissue sample suspected of having a heterodimer of the Bcl-2
family, (ii) contacting the sample with the antibody, (iii)
detecting a signal indicative of binding of the antibody to the
heterodimer, and (iv) determining the presence of the heterodimer
in the sample based on the intensity of the signal. Examples of the
heterodimer include Bim/Mcl-1 and Bim/Bcl-2. The tissue sample
examined in this method can be a peripheral blood sample, a
lymph-node sample, a bond marrow sample, or an organ tissue sample.
Preferably, it is a mitochondrial fraction.
[0007] Also within the scope of this invention is a method for
assessing whether a patient is sensitive or resistance to a drug
that interferes with formation of a heterodimer of the Bcl-2 family
based on the presence of that heterodimer in the patient. A cancer
patient is sensitive to an apoptosis inducer that blocks formation
of an anti-apoptotic Bcl-2 heterodimer if this heterodimer is
present in that patient. A neurodegenerative disease or
cardiovascular disease patient, on the other hand, is responsive to
an apoptosis inhibitor that blocks formation of a pro-apoptotic
Bcl-2 heterodimer if this heterodimer is present in that
patient.
[0008] The details of one or more examples of the invention are set
forth in the description below. Other features or advantages of the
present invention will be apparent from the following drawings,
detailed description of several examples, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The drawings are first described.
[0010] FIG. 1 is a schematic illustration depicting the
conformational change of a multidomain Bcl-2 protein induced by
dimerization with a BH3-only Bcl-2 protein.
[0011] FIG. 2 is a schematic illustration depicting the process of
selecting antibodies specific to Bcl-2 heterodimers via an
immunoassay. Panel A: antibodies binding to a Bcl-2 heterodimer
being positively selected. Panel B: antibodies binding to
non-dimerized members of the heterodimer being negatively selected.
Panel C: illustration of what the symbols in Panels A and B
represent.
[0012] FIG. 3 is a schematic illustration depicting an immunoassay
for profiling Bcl-2 heterodimers on mitochondria using the antibody
of this invention, i.e., an antibody specifically recognizes Bcl-2
heterodimers, and an anti-VDAC antibody that recognizes
mitochondria. Panel A: mitochondria presenting Bcl-2 heterodimers
being recognized by both the antibody of this invention and an
anti-VDAC antibody. Panel B: mitochondria not presenting Bcl-2
heterodimers being recognized by only the anti-VDAC antibody. Panel
C: illustration of what the symbols in Panels A and B
represent.
[0013] FIG. 4 is a schematic illustration showing identification of
scFv antibodies specific to Bcl-2 heterodimers from a yeast display
scFv library.
[0014] FIG. 5 is a schematic illustration depicting a Luminex bead
assay for profiling Bcl-2 heterodimers in a patient sample. Panel
A: multi-color beads used in the Luminex bead assay. Panel B:
illustration of what the symbols in Panels A and B represent.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Disclosed herein is an isolated antibody specific to a Bcl-2
heterodimer, i.e., a naturally-occurring heterodimer formed between
two Bcl-2 proteins.
[0016] Bcl-2 proteins, found in mitochondria, are major regulators
of the commitment to programmed cell death and executioners of
death/survival signals. See Reed, Natural Clinical Practice
Oncology, 3:388-398 (2006), Green et al., Cancer Cell 1:19-30
(2002), and Adams et al., Cold Spring Harb. Symp. Quant. Biol.
70:469-477 (2005). There are four subgroups of Bcl-2 proteins: (i)
multi-domain anti-apoptotic Bcl-2 proteins, (ii) multi-domain
pro-apoptotic Bcl-2 proteins, (iii) activator BH3-only Bcl-2
proteins, and (iv) sensitizer BH3-only Bcl-2 proteins. Table 1
below lists major human Bcl-2 proteins and their GenBank accession
numbers:
TABLE-US-00001 TABLE 1 Human Bcl-2 Proteins Bcl-2 Proteins GenBank
Accession Numbers Multi-domain Bcl-2 AAH27258 (Jul. 15, 2006)
Anti-Apoptotic Bcl-XL AAH19307 (Jul. 15, 2006) Bcl-2 Proteins Mcl-1
AAF64255 (Jul. 17, 2000) BCL-w AAB09055 (Sep. 29, 1996) BFL-1
Q16548 (Mar. 3, 2009) Multi-domain BAX Q07812 (Apr. 14, 2009)
Pro-Apoptotic BAK Q16611 (Apr. 14, 2009) Bcl-2 Proteins Sensitizer
BH3- BAD CAG46757 (Jun. 29, 2004) only Bcl-2 BIK CAG30276 (Oct. 16,
2008) Proteins NOXA Q13794 (Mar. 3, 2009) HRK AAC34931 (Sep. 9,
1998) BMF AAH69328 (Aug. 19, 2004); AAH60783 (Jan. 27, 2004) PUMA
Q9BXH1 (Apr. 14, 2009) Mule Q7Z6Z7 (Apr. 14, 2009) Activator BH3-
BID P55957 (Mar. 3, 2009) only Bcl-2 BIM O43521 (Apr. 14, 2009)
Proteins
Other Bcl-2 proteins, if any, can be identified by homologous
search using the amino acid sequence of a known Bcl-2 protein as a
query.
[0017] It is known that members in one subgroup of Bcl-2 proteins
form heterodimers with members in a different subgroup to regulate
apoptosis. Formation of a heterodimer induces conformational
changes in both members of the heterodimer, resulting in exposure
of antigenic epitopes that are sequestered in both members before
dimerization. See FIG. 1. The isolated antibody of this invention
specifically recognizes such an epitope (e.g., the arrow epitope
shown in FIG. 1). In other words, it only binds to a heterodimer of
the Bcl-2 family, not to either non-dimerized member.
[0018] This antibody can be a whole immunoglobulin or a fragment
thereof that retains antigen-binding activity. It can be a
genetically modified immunoglobulin, including scFv antibody,
chimeric antibody, and humanized antibody. The term "isolated
antibody" used herein refers to an antibody substantially free from
naturally associated molecules, i.e., the naturally associated
molecules constituting at most 20% by dry weight of a preparation
containing the antibody.
[0019] The antibody of this invention can be prepared by
conventional methods. See, for example, Harlow and Lane, (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York. In general, a heterodimer of the Bcl-2 family can be prepared
by producing its two members separately by recombinant technology
and then incubate both members under suitable conditions to allow
formation of the heterodimer. To produce antibodies against the
heterodimer, the heterodimer, optionally coupled to a carrier
protein (e.g., KLH) can be mixed with an adjuvant, and injected
into a host animal. Antibodies produced in the animal can then be
purified by heterodimer affinity chromatography. Commonly employed
host animals include rabbits, mice, guinea pigs, and rats. Various
adjuvants that can be used to increase the immunological response
depend on the host species and include Freund's adjuvant (complete
and incomplete), mineral gels such as aluminum hydroxide, CpG,
surface-active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. Useful human adjuvants include BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies,
i.e., heterogeneous populations of antibody molecules, are present
in the sera of the immunized animal.
[0020] Monoclonal antibodies, i.e., homogeneous populations of
antibody molecules, can be prepared using standard hybridoma
technology (see, for example, Kohler et al. (1975) Nature 256, 495;
Kohler et al. (1976) Eur. J. Immunol. 6, 511; Kohler et al. (1976)
Eur J Immunol 6, 292; and Hammerling et al. (1981) Monoclonal
Antibodies and T Cell Hybridomas, Elsevier, N.Y.). In particular,
monoclonal antibodies can be obtained by any technique that
provides for the production of antibody molecules by continuous
cell lines in culture such as described in Kohler et al. (1975)
Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cell
hybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole
et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026, and the
EBV-hybridoma technique (Cole et al. (1983) Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies
can be of any immunoglobulin class including IgG, IgM, IgE, IgA,
IgD, and any subclass thereof. The hybridoma producing the
monoclonal antibodies of the invention may be cultivated in vitro
or in vivo. The ability to produce high titers of monoclonal
antibodies in vivo makes it a particularly useful method of
production.
[0021] In addition, techniques developed for the production of
"chimeric antibodies" can be used. See, e.g., Morrison et al.
(1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984)
Nature 312, 604; and Takeda et al. (1984) nature 314:452. A
chimeric antibody is a molecule in which different portions are
derived from different animal species, such as those having a
variable region derived from a murine monoclonal antibody and a
human immunoglobulin constant region. Alternatively, techniques
described for the production of single chain antibodies (U.S. Pat.
Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or
yeast library of scFv antibodies. scFv antibodies are formed by
linking the heavy and light chain fragments of the Fv region via an
amino acid bridge.
[0022] Moreover, antibody fragments can be generated by known
techniques. For example, such fragments include, but are not
limited to, F(ab').sub.2 fragments that can be produced by pepsin
digestion of an antibody molecule, and Fab fragments that can be
generated by reducing the disulfide bridges of F(ab').sub.2
fragments. Antibodies can also be humanized by methods known in the
art. For example, monoclonal antibodies with a desired binding
specificity can be commercially humanized (Scotgene, Scotland; and
Oxford Molecular, Palo Alto, Calif.). Fully human antibodies, such
as those expressed in transgenic animals are also features of the
invention (see, e.g., Green et al. (1994) Nature Genetics 7, 13;
and U.S. Pat. Nos. 5,545,806 and 5,569,825).
[0023] The antibodies prepared by any of the methods described
above are confirmed for their binding to a Bcl-2 heterodimer. They
are further subjected to a negative selection to exclude those that
also bind to either non-dimerized member of the heterodimer. See
FIG. 2. One example follows. Each of the two members, i.e., monomer
A and monomer B, is labeled with a distinct fluorescent dye, i.e.,
dye x and dye y, respectively. Dyes x and y have different optimal
emission wavelengths. The antibody is first incubated with labeled
monomer A, labeled monomer B, or the A/B heterodimer (double
labeled) for a suitable period and then captured by GamaBind
Sepharose beads. Whether the antibody is capable of binding to
either monomer or to the heterodimer can be determined based on the
fluorescent signal released from the captured antibody. Antibodies
that bind to the heterodimer and not to either non-dimerized member
are selected See FIG. 2.
[0024] The antibody of this invention can be used to detect the
presence or absence of a Bcl-2 heterodimer in a patient sample,
particularly, a fixed tissue sample or a mitochondrial fraction, by
conventional methods, e.g., histochemistry staining. See FIG. 3. In
one example, a plurality of antibodies specific to various Bcl-2
heterodimers are used to profile the presence of particular Bcl-2
heterodimers on the outer membranes of mitochondria in multiple
tissue samples from a patient. Tissues at various disease stages
(e.g., malignancy stages) can be collected from the same patient.
Mitochondrial fractions can be prepared from these tissues and,
using a plurality of the antibodies of this invention, the
fractions can be profiled for the presence/absence of Bcl-2
heterodimers.
[0025] Also disclosed herein is a method of predicting a human
patient's responsiveness to a drug that interferes with formation
of a particular Bcl-2 heterodimer, directly or indirectly, based on
the presence of that Bcl-2 heterodimer in the patient.
[0026] It well known that Bcl-2 proteins paly an essential role in
regulating apoptosis via formation of heterodimers between members
in different Bcl-2 sub-groups. See Table 1 above. An activator
BH3-only Bcl-2 protein (i.e., BID or BIM) binds to a multi-domain
pro-apoptotic Bcl-2 protein (i.e., BAX or BAK), triggering
mitochondrial outer membrane permeabilization (MOMP), which leads
to cell death. A multi-domain anti-apoptotic Bcl-2 protein (e.g.,
Bcl-2 or Mcl-1) can bind to BAX and BAK, and also sequester an
activator BH3-only protein from binding to BAX or BAK.
Consequently, it blocks the MOMP process, resulting in cell
survival. The activity of a multi-domain anti-apoptotic Bcl-2
protein is regulated by the sensitizer BH3-only proteins. This
subgroup of Bcl-2 proteins promotes apoptosis by binding to the
anti-apoptotic Bcl-2 protein, displacing the activator BH3-only
Bcl-2 proteins so that they are released to bind to the
pro-apoptotic Bcl-2 proteins, thereby triggering the MOMP process.
In short, there are two types of Bcl-2 heterodimers: (1)
pro-apoptotic Bcl-2 heterodimers, formed between an activator
BH3-only Bcl-2 protein and a multi-domain pro-apoptotic Bcl-2
protein; and (2) anti-apoptotic Bcl-2 heterodimers, formed between
a multi-domain anti-apoptotic Bcl-2 protein and an activator
BH3-only Bcl-2 protein or between a multi-domain anti-apoptotic
Bcl-2 protein and a multi-domain pro-apoptotic Bcl-2 protein.
Formation of pro-apoptotic Bcl-2 heterodimers promote apoptosis
while formation of anti-apoptotic heterodimers promote cell
survival.
[0027] The presence of a particular pro- or anti-apoptotic Bcl-2
heterodimer in a patient is known to indicate that patient's
responsiveness to a drug that blocks formation of the particular
heterodimer and inhibits its function. See, e.g., Letai, Nature
Reviews Cancer, 8:121-132 (2008). In one example, the drug is a
mimetic of BH3-only protein that competes against the BH3-only
protein for binding to its cognate partner. In another example, the
drug targets an upstream apoptotic factor and ultimately blocks
formation of a Bcl-2 heterodimer.
[0028] Many cancer drugs induce apoptosis in cancer cells by
blocking formation of anti-apoptotic Bcl-2 heterodimers. The
presence of a particular anti-apoptotic Bcl-2 heterodimer in a
cancer patient indicates that this patient is sensitive to a drug
that interferes with formation of this anti-apoptotic Bcl-2
heterodimer. See Deng, et al Cancer Cell 12(2):171-85 (2007). On
the other hand, apoptosis inhibitors are used for treating
neurodegenerative disease or cardiovascular disease, both of which
involve apoptosis. In these cases, the presence of a particular
pro-apoptotic Bcl-2 heterodimer in a neurodegenerative disease
patient or a cardiovascular disease patient indicates that such a
patient is sensitive to an apoptosis inhibitor that blocks
formation of the particular pro-apoptotic Bcl-2 heterodimer.
[0029] Bcl-2 heterodimer profiling can also be used to predict
responsiveness to drugs targeting the apoptotic pathway in patients
suffering from other apoptosis-related diseases, e.g., autoimmune
disease (see Adams et al., Cold Spring Harb Symp Quant Biol.
70:469-477; 2005).
[0030] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference.
EXAMPLE 1
Preparation of Monoclonal Antibodies Specific to Bcl-2
Heterodimers
[0031] Genes encoding human Bcl-xL, Bcl-2, and Mcl-2 are cloned and
mutated to delete their transmembrane domains. The mutated genes
are linked to a nucleotide sequence encoding
glutathione-S-transferase (GST) and cloned into pGEX 4T-1 to obtain
DNA constructs for expressing Bcl-xL(.DELTA.td)-GST,
Bcl-2(.DELTA.td)-GST, and Mcl-2(.DELTA.td)-GST fusion proteins. DNA
constructs for expressing full length human Bax, Bak, Bak, Bim,
Bid, Bad, Puma, and Noxa, all fused with GST, are prepared by
recombinant technology.
[0032] All of the DNA constructs are introduced into BL2I E. coli
cells. Positive transformants are cultured in a suitable medium and
expression of the fusion proteins are induced with
isopropyl-1-thio-.beta.-D-galactopyranoside. The expressed fusion
proteins are purified using Amersham Hitrap Glutathion e column on
the ACTA-FPLC (Amersham) and accurately quantified using
spectrophotometry.
[0033] Bcl-xL(.DELTA.td)-GST, Bcl-2(.DELTA.td)-GST, or
Mcl-2(.DELTA.td)-GST is mixed with Bax-GST, Bak-GST, Bak-GST,
Bim-GST, Bid-GST, Bad-GST, Puma-GST, or Noxa-GST at equamolar
amounts in PBS. The mixture is stirred on ice for 12 hours to allow
formation of heterodimers.
The heterodimmers are purified using a sepharose 12 column
(Pharmacia) on a ACTA-FPLC (Amersham), following the method
described in Zue et al., Protein Science 6: 781-788 (2007).
[0034] Each of the heterodimers (1 .mu.g) is suspended in
monophosphoryl lipid A plus trehalose dicorynomycloate adjuvant
(Ribi Immunochem. Research Inc., Hamilton, Mont.). The mixture thus
formed are injected into Balb/c mice at each hind foot pad once
every 3-4 days for 14 times. Three days after the final injection,
spleen cells are removed from the mice and a single cell suspension
is prepared in a DMEM medium (Gibco/BRL Corp.) supplemented with 1%
penicillin-streptomycin. The spleen cells are fused with murine
myeloma cells P3X63AgU.1 (ATCC CRL 1597) using 35% polyethylene
glycol and cultured in 96-well culture plates.
[0035] Hybridomas are selected in super DMEM [DMEM supplemented
with 10% fetal calf serum FCS, 100 mM pyruvate, 100 U/ml insulin,
100 mM oxaloacetic acid 2 mM glutamine, 1% nonessential amino acids
(GIBCO/BRL), 100 U/ml penicillin, and 100 .mu.g/ml streptomycin]
containing 100 .mu.M hypoxanthine, 0.4 .mu.M aminopterin, and 16
.mu.M thymidine (HAT), (Sigma Chemical Co., St. Louis. Mo.).
[0036] Hybridoma cells are fed with 200 .mu.l of super DMEM
containing 10% FCS and antibiotics. Ten days after the fusion,
supernatants of the hybridoma cultures are collected and screened
for the presence of antibodies that bind to the cognate heterodimer
protein and/or to either member of the heterodimer (as negative
controls) in a capture ELISA as described in Certo et al., Cancer
Cell., 9(5):351-365 (2006).
[0037] Briefly, 96-well microtiter plates (Maxisorb; Nunc,
Kamstrup, Denmark) are coated with 50 .mu.l (1 .mu.g/ml) of a
heterodimer or a member of the heterodimer at 4.degree. C.
overnight. The plates are then washed three times with PBS
containing 0.05% TWEEN 20.TM. (PBST) and blocked with 50 .mu.l PBS
containing 2.0% bovine serum albumin (BSA) at room temperature for
1 hour. The plates are then washed again three times with PBST.
Afterwards, 100 .mu.l of a hybridoma supernatant is added to
designated wells. The plates are incubated at room temperature for
1 hour on a shaker apparatus and then washed three times with wash
buffer. Next, 50 .mu.l HRP-conjugated goat anti-mouse IgG Fe
(Cappel Laboratories), diluted 1:1000 in assay buffer (0.5% bovine
serum albumin, 0.05% % TWEEN 20.TM., 0.01% Thimersol in PBS), is
added to each well. The plates are then incubated for 1 hour at
room temperature on a shaker apparatus and washed three times with
wash buffer, followed by addition of 50 .mu.l of substrate DACO and
incubation at room temperature for 10 minutes. 50 .mu.l diethyl
glycol were added to each well to stop the reaction and absorbance
at 450 nm in each well is read in a microtiter plate reader.
[0038] Hybridoma cells producing antibodies that bind to a
heterodimer but not to either member of the heterodimer are
selected. These positive hybridoma cells are cloned twice and the
specificity of the antibodies produced thereby are retested. The
isotypes of the antibodies having the desired specificity are
determined by conventional methods, e.g., using isotype specific
goat anti-mouse Igs (Fisher Biotech, Pittsburgh, Pa.).
EXAMPLE 2
Preparation of Polyclonal Antibodies Specific to Bcl-2
Heterodimers
[0039] New Zealand rabbits were immunized on the back and proximal
limbs of the rabbits with 0.1 ml of a Bcl-2 heterodimer (50 ug/ml)
prepared following the method described in Example 1. The
heterodimer is pre-mixed with 50% Freund's complete adjuvant. The
immunization is repeated 28th days later. On day 35, 0.5 ml of
blood is obtained from each of the immunized rabbits and antibody
titers in the blood samples are determined by ELISA. Anti-sera are
collected from the arterial carotid of rabbits having high antibody
titers.
[0040] The specificity of the antibodies in each antiserum is
examined by conventional methods, e.g., the immunoprecipitation and
FACS assays described in Examples 4 and 5 below.
EXAMPLE 3
Screening for scFv Antibodies Specific to Bcl-2 heterodimers Using
a Yeast scFv Library
[0041] A nonimmune human scFv yeast library (using expression
vector pYD1) is obtained from Pacific Northwest National
Laboratories (http://www.sysbio.org/dataresources/singlechain.stm).
In this library, a scFv antibody, in which the heavy and light
chains is connected by a flexible polypeptide linker, is fused to
the adhesion subunit of the yeast agglutinin protein Aga2p and the
HA-tag. Upon expression, the scFv is located on the surface of a
yeast host cell via binding of Aga2P to Aga1P, a cell surface
protein. See FIG. 4. Each yeast cell typically displays
1.times.10.sup.5 to 1.times.10.sup.6 copies of the scFv and the
surface expression of the scFv. Variations in surface expression
can be measured through immunofluorescence labeling of the HA-tag
flanking the scFv region. See FIG. 4.
[0042] The scFv library described above is introduced into yeast
strain EBY100 (Invitrogen) and scFv antibodies having the desired
specificity are identified as follows. The EBY yeast cells are
first grown overnight in 1 liter of SDCAA medium (containing 20 g
dextrose, 6.7 g Difco yeast nitrogen base, 5 g Bacto casamino
acids, 5.4 g Na.sub.2HPO.sub.4 and 8.56 g
NaH.sub.2PO.sub.4H.sub.2O). 1.times.10.sup.10 yeast cells from the
overnight culture are precipitated by centrifugation at 2,500 g for
5 minutes and resuspended in SGCAA medium (a medium identical to
SDACC except that it contains galactose instead of dextrose) to an
absorbance of about 0.5-1 at 600 nm. The yeast cells are then
cultured at 20.degree. C. for 36 h to allow expression of scFv
antibodies. Afterwards, the cells are collected by centrifugation
at 2,500 g for 5 min. The cell pellet is washed with 25 ml PBS.
[0043] Yeast cells expressing scFv antibodies are sorted by flow
cytometry. Briefly, about 1.times.10.sup.6 to 1.times.10.sup.7
yeast cells prepared as described above are collected via
centrifugation at 14,000 g for 30 seconds, washed with 1 ml PBS
buffer, and mixed with 2 .mu.l of 10 .mu.g/ml anti-HA phycoerythrin
monoclonal antibody (SIGMA-ALDRICH) and Bcl-2/Bid heterodimer, in
which Bcl-2 is labeled with FITC and Bid is labeled with Texas red.
After being incubated at room temperature for 1 hour, the mixture
is centrifuged at 12,000 g for 30 seconds to precipitate yeast
cells. The cell pellet thus formed is resuspended in 500 .mu.l 10
mM Tris (final cell density .about.10.sup.6/ml) and subjected to
cell sorting by flow cytometry as follows.
[0044] A flow cytometry protocol is pre-determined using EBY100
yeast cells mixed with the anti-HA phycoerythrin antibody as a
positive control and EBY100 yeast cells mixed with the
double-labeled heterodimer as a negative control. Compensation is
performed to reject crosstalk between the FITC, Texas red, and
phycoerythrin channels of the fluorescence detector. The labeled
yeast cells are loaded into a FACSAria Cell-Sorter (Becton
Dickinson, Mountain View, Calif.) and gated on forward- and side
scatter channels. An appropriate sort gate in the FITC/Texas
red/phycoerythrin positive quadrant is drawn and the top 5%
triple-positive yeast cells are collected in 1 ml SDCAA media. If
necessary, the top 0.1% triple-positive yeast cells are collected
to ensure that only cells having high affinity to Bcl-2/Bid
heterodimer is sorted.
[0045] The triple-positive cells thus identified are suspended in
10 ml SDCAA and grown over night at 30.degree. C. These cells are
then subjected to two rounds of negative selection to exclude cells
expressing scFv antibodies that also bind to Bcl-2 or Bid monomer.
More specifically, the cells are incubated with FITC-labeled Bcl-2
and Texas red-labeled Bid and following the same procedure
described above, FITC and Texas red double negative cells are
sorted. The cells thus collected are labeled with the
double-labeled Bcl-2/Bid heterodimer to confirm their binding to
the heterodimer.
[0046] The yeast cell thus identified are diluted and plated to
allow formation of individual clones, Plasmid DNAs are isolated
from these clones using a Zymoprep kit (Zymo Research, Orange,
Calif.) as described in Weaver-Feldhaus et al., Protein
Engineering, Design & Selection vol. 18, no. 11, pp 527-536
(2005). The scFv sequence included in each plasmid DNA is
determined following the method described in Chao et al., Nature
Protocols 1:755-768 (2006).
[0047] The scFv antibodies thus identified are analyzed by ELISA
and FACS to confirm their specificity to Bcl-2/Bid heterodimer.
They can subject to mutagenesis to select for scFv antibodies
having higher affinity and specificity to Bcl-2/Bid
heterodimer.
EXAMPLE 4
Select Antibodies Specific to Bcl-2 Heterodimers by
Immunoprecipitation
[0048] An immunoprecipitation assay, as illustrated in FIG. 2, is
performed to ensure that the antibodies obtained in Example 1 above
are specific to Bcl-2 heterodimers. The Two members of a Bcl-2
heterodimer are conjugated with two fluorescent probes that have
distinct emission spectra, i.e., one labeled with fluorescein
isothiocyanate (FITC; which emit at 488 nm) and the other labeled
with Texas red (which emits at 590 nm). See FIG. XX. The labeled
members are incubated together to allow formation of the Bcl-2
heterodimer, following the method described in Example 1 above. 0.1
.mu.g of the heterodimer thus formed is incubated with 50 uL of
supernatant from a hybridoma clone that produces an antibody of
interest in 0.5 mL PBS containing 0.05% tween-20. The non-dimerized
labeled members of the heterodimer are used as negative controls.
The mixtures are incubated for 1 hour on ice to allow formation of
antibody-antigen complexes and 10 ul of GammaBing-G sepharose beds
(GE Healthcare Piscataway, N.Y.) are added to the mixture. After
being incubated on ice for 30 minutes on ice with rotation, the
mixtures are centrifuged at 10,000 g for 30 seconds. The pellet
beads, to which the antibody-antigen complexes are attached, are
washed several times and measured for optical density at 488 nm
(OD.sub.488) and 590 nm (OD.sub.590). The specificity of the
antibody is determined based on the values of OD.sub.488 and 590 nm
OD.sub.590.
EXAMPLE 5
Select Antibodies Specific to Bcl-2/Bim Heterodimer by Flow
Cytometry
[0049] Mitochondria from cells having a high level of Bcl2/Bim
heterodimer and from cells having Bcl-2 knocked-out are purified
and labeled as described in Methods Enzymol. 2000; 322:235-42 and
Cancer Cell 2004; Cell 2, 183-192. See FIG. 3, Panels A and B.
Briefly, the just-mentioned two types of cells are suspended in
cold hypotonic lysis buffer, 259 mM sucrose, 10 mM Tris-Hcl pH
(7.4), 1 mM EGTA, and homogenized in a dounce rotary Teflon pestle,
followed by 6-10 expulsions through a 27-gauge needle. The
fractions containing mitochondria are collected by differential
centrifugation and resuspended in an assay binding buffer (125 mM
KCL, 10 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, pH 7.2, 20 uM ATP) to a
final protein concentration of 5 mg/ml.
[0050] Twenty-five microliters of the suspension (4 mg of
mitochondrial protein/ml) are suspended with PBS containing 1% FCS,
(FACS buffer) and mixed with 100 .mu.l of a hybridoma culture
supernatant containing a test antibody or a test antibody in
purified form (purified on Protein-G sepharose column; 10 .mu.g/ml
in the FACS buffer) in a U-bottom microtiter wells.
Rhodamine-labeled polyclonal anti-VDAC-1 antibody (Merridian Life
Sciences, Inc. Cincinnati, Ohio), an antibody specific to
mitochondria, is also added to each well. 100 .mu.l of fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin are
then added to the mitochondria-antibody mixture. After being
incubated at 4.degree. C. for 30 min, the mixture is washed with
the FACS buffer and centrifuged at 12 g for 5 minutes to
precipitate mitochondria The mitochondrial pellet is resuspended in
150 .mu.l of FACS buffer and analyzed by FACScan (Becton Dickinson,
Mountain View, Calif.), with flow cytometry parameters
pre-determined using control mitochondrial samples, i.e., unlabeled
mitochondria as a negative control sample and anti-VDAC-1-Rhodamine
labeled mitochondria as a positive control. The mitochondrial
suspension is loaded into the flow cytometry apparatus using a FACS
tube and signals released from FITC and Rhodamine are detected. If
the mitochondrial suspension is double positive for both FITC and
Rhodamine, it indicates that the test antibody is capable of
binding to the Bcl-2/Bim heterodimer. See FIG. 3, Panel A.
EXAMPLE 6
Detecting Bcl-2 Heterodimers in Fixed Cells
[0051] Cells characterized for having a prevalent Mcl-1/Bim or
Bcl-2/Bid heterodimer are used in this study. See Certo et al.,
Cancer Cell. 9(5):351-365 (2006), Moore et al., J. Clin. Invest.
117(1):112-121 (2007), Deng et al., Cancer Cell. 12(2):171-185
(2007), and Letai, Nature Reviews Cancer 8:121 (2008). These cells,
placed on cover slips, are fixed with 2-4% formaldehyde
(Formaldehyde, 16%, methanol free, Polysciences, Inc.) in PBS for
15 minutes at room temperature. The cell-containing cover slips are
rinsed with PBS three times, 5 minutes for each. the slips are then
soaked in a blocking buffer (TBST/5% normal goat serum: to 5 ml
1.times.TBST add 250 .mu.l normal goat serum) for 60 minutes. After
the blocking buffer is aspirated, an antibody specific to either
Mcl-1/Bim or Bcl-2/Bid heterodimer (0.1 to 15 mg/ml) is added to
the slips. An anti-human-VDAC-1 antibody is also added to localize
mitochondria. After being incubated at 4.degree. C. overnight, the
slips are rinsed three times with PBS, 5 minutes each time. A
fluorochrome-conjugated secondary antibody, diluted in a dilution
buffer, is then added. After being incubated for 1-2 hours at room
temperature in dark, the slips are rinsed with PBS three time, 2
minutes each time, and subsequently treated with Prolong.RTM. Gold
Antifade Reagent (Invitrogen, San Diego, Calif.). The slips are
then sealed by painting around edges of the slips with nail polish
and observed under an inverted fluorescent microscope. Localization
of the antibody on mitochondria indicates that the antibody
recognizes Mcl-1/Bim heterodimer or Bcl-2/Bid heterodimer.
EXAMPLE 7
Detecting Bcl-2 Heterodimers in Fixed Tissue Samples
[0052] Paraffin embedded and frozen thin section tissue samples
from cancer patients and healthy subjects are purchased from
Imgenex, San Diego, Calif. These samples are spotted on microarray
chips (4 mm.times.4 mm spots that are 4 mm thick). The adjacent
normal tissues from the same patients/healthy subjects are also
spotted on the array chips.
[0053] The microarray chips mentioned above are washed in turn with
xylene three times, 5 minutes each time, 100% ethanol twice, 10
minutes each time, 95% ethanol, twice, 10 minutes each time, and
finally dH.sub.2O twice, 5 minutes each time. The chips are then
soaked in 1 mM EDTA, pH 8.0, heated to boiling, and then kept at a
sub-boiling temperature for 15 minutes.
[0054] If the tissue samples on the microarray chips are fixed with
formalin, the chips are washed in turn with 100%, 95%, 80% ethanol
3 times each, 3 minutes each time, followed by two washes with
dH.sub.2O, 3 minutes each. The chips are then soaked in 0.01 M
sodium citrate, pH 6.0 for 20 minutes.
[0055] The chips are then washed with dH.sub.2O three times, 5
minutes each time, incubated in 3% hydrogen peroxide for 10 minutes
(this step is not needed for formalin fixed samples), and washed
again with dH.sub.2O twice, 5 minutes each time.
[0056] Next, the chips are subjected to immunostaining using the
antibodies prepared in Example 1 or an anti-VDAC1 antibody as a
control. The chips are soaked in a wash buffer for 5 minutes and
then in 100-400 .mu.l of a blocking buffer (TBST containing 5%
normal goat serum) for one hour. After decanting the blocking
solution, the chips are incubated with 100-400 .mu.l of an
anti-Bcl-2-heterodimer antibody (primary antibody), diluted to 0.1
to 15 ug/ml for each chip, overnight at 4.degree. C. Afterwards the
chips are washed with the wash buffer three times, 5 minutes each
time, and then incubated with 100-400 .mu.l of a biotinylated goat
anti-mouse Ig antibody (the secondary antibody), which is diluted
in TBST following the manufacturer's protocol, for 30 minutes at
room temperature. The chips are then washed with the wash buffer
three times, 5 minutes each time, and incubated with 100-400 .mu.l
ABC reagent (Vectastain ABC Kit, Vector Laboratories, Inc.,
Burlingame, Calif.), which is prepared following the manufacturer's
instructions, for 30 minutes at room temperature. After being
washed for three times with the wash buffer, the chips are
incubated with 100-400 .mu.l DAB for signal development. The chips
are immersed in dH.sub.2O immediately after a color has developed
thereon. When necessary, the chips are counterstained with
hematoxylin and DAPI following manufacturer's instructions.
The stained chips are dehydrated by incubation sequentially in 95%
ethanol two times, 10 seconds each, in 100% ethanol two times, 10
seconds each, and finally in xylene two times, 10 seconds each. The
chips are then mounted with cover slips and examined using
Fluorescence and UV microscopy for staining patterns. The staining
patterns obtained from cancer tissue samples are compared with
those obtained from adjacent normal tissues.
EXAMPLE 8
Profiling Bcl-2 Heterodimers on Mitochondria Using Color Coded
Micro-Beads
[0057] As shown in FIG. 5, micro-beads of 5 micron diameter and
color coded (Luminex Incorporated) are derivatized with an
anti-human VDAC-1 antibody, which recognizes mitochondria.
Mitochondrial samples prepared by different patients, as described
above, are incubated with different colored VDAC derivatized
Luminex beads and fluorescent probe-labeled antibodies specific to
Bcl-2 heterodimers, as prepared by the method described in Example
1. The labeling pattern (i.e., color combination) of each
mitochondrial sample is examined by conventional methods and the
Bcl-2 heterodimer profile of each patient is determined based on
the labeling patter. This profile can be used to diagnose certain
disease indications and chemsensitivities, as well as predict
outcomes of treatment for cancer or other diseases associated with
apoptosis.
OTHER EMBODIMENTS
[0058] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0059] From the above description, on skilled in the art can easily
ascertain the essential characteristics of the present invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions. Thus, other embodiments are also
within the claims.
* * * * *
References